Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability

Significance

During transcription, the mRNA may hybridize back with its template DNA, forming a structure called R loop. These structures have been associated with genome instability and human disease. Using budding yeast as a model organism to screen for new genes preventing R loops, we identified MLP1 and subsequently showed that the nuclear basket protein Mlp1/2 has a role in preventing R loop formation and genome instability in yeast. Our work indicates that R loops are formed in the nucleoplasm and that proximity of transcribed chromatin to the nuclear pore constrains R loop formation. Our study opens additional perspectives to understand the role of RNA in the control of genome integrity as a function of nuclear location.

Abstract

During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA–RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1∆ cells. The same occurred in THO-deficient hpr1∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.

During transcription elongation, the nascent mRNA may hybridize back with the DNA template forming a DNA–RNA hybrid and a displaced ssDNA, a structure called R loop. Although R loops can occur naturally as a physiologically relevant intermediate in specific processes, pathological R loops was revealed as a major determinant of genome instability (1, 2). Different protein complexes that act all along the path from the transcription site to the nuclear pore complex (NPC) enable the coordination of messenger ribonucleoprotein particle (mRNP) biogenesis and export. THO is an evolutionary conserved complex composed of the Tho2, Hpr1, Mft1, and Thp2 proteins in yeast (3), which associates with mRNA export factors such as Sub2/UAP56 (4). Mutations in THO components not only impair transcription but also lead to mRNA export defects and transcription-dependent genomic instability in yeast and metazoans (3, 5–7) that is a consequence of the accumulation of cotranscriptional R loops (8). Such unscheduled R loops interfere with DNA replication (9, 10), ultimately leading to genomic instability. Together with THO, a growing number of factors working at different stages of mRNP biogenesis and export have been shown to prevent R loops and contribute to maintaining genome stability (11–14), establishing mRNP biogenesis as one of the cellular mechanism that controls R loop formation in eukaryotes. Other mechanisms include removal of negative supercoiling by topoisomerase I; degradation of the RNA moiety by the catalytic activity of RNase H1; unwinding of the DNA–RNA hybrids by helicases such as Sen1/Senataxin, DDX19, DDX23, and Pif1; or nucleosomes as a barrier to R loop formation (15–23). In addition, a number of replisome-associated proteins contribute to the handling of R loops, such as Fanconi anemia/BRCA factors or ssDNA binding protein RPA (24–28).

With the aim at understanding the mechanisms that control R loop formation, we used the activation-induced cytidine deaminase (AID) that targets the ssDNA formed at R loops (29) to screen a selection of 400 viable yeast strains deleted of genes with nuclear functions for AID-dependent hyperrecombination. This allowed us to identify MLP1 as a gene involved in preventing R loop accumulation. The yeast myosin-like protein 1 and 2 (Mlp1 and Mlp2) are structural components of the nuclear pore basket that form fibers anchored at the NPC (30). Mlp1 physically interacts with the mRNP component Nab2 (31), and Mlp1/2 have been proposed to be a docking site for mRNPs during export and to participate in mRNP quality control (32, 33). Gene gating, the transient localization of transcribed DNA in the proximity of the NPC has been proposed to facilitate the formation of an export-competent mRNP (34). Consistently, Mlp1/2 proteins preferentially associate with highly transcribed genes in an RNA-dependent manner (35), and Mlp1 is also required for the docking of actively transcribed DNA to the NPC (36). However, gene gating may also increase the torsional stress, enhancing the transcriptional barrier to replication, an effect counteracted by intra-S checkpoint activation by a mechanism involving phosphorylation of Mlp1 (37). Our study shows that loss of Mlp1 and/or Mlp2 leads to R loop accumulation, genome instability, and replication impairment, phenotypes that can be reverted by RNase H1 overexpression, pointing toward R loops as the causative event. Importantly, R loops are suppressed in mlp1∆ and hpr1∆ cells by artificially tethering a locus of interest to the NPC, indicating that physical proximity to the NPC is sufficient to prevent R loop accumulation. We conclude that gene gating prevents R loop formation.

Results

Screening of Yeast Null Mutants for AID-Mediated Hyperrecombination.

We previously showed that expression of AID, a B-cell enzyme essential for somatic hypermutation and class switch recombination, in yeast THO mutants exacerbate their transcription-dependent hyperrecombination phenotype (29). Here, we used AID overexpression as a tool to uncover mutations that lead to R loop accumulation in Saccharomyces cerevisiae. Using gene ontology (GO) term annotations, we selected 400 viable deletion strains lacking genes with nuclear functions from the yeast knockout collection (Table S1). The 400 strains were transformed with plasmid p313LZGAID, carrying a direct-repeat recombination construct with the lacZ gene between two leu2 repeats and expressing the human AID gene under the control of the GAL1 promoter. The transformed cells, including wild-type, hpr1∆ and mft1∆, were grown in galactose to induce AID expression and plated on appropriate media to assess the amount of recombinants (Fig. S1A). The 123 candidates with high levels of recombination in AID-expression conditions in at least two of three independent experiments were selected for further analysis. Next, four fresh transformants were grown either in glucose (−AID) or galactose (+AID), and serial dilutions were plated on selective media for each candidate (Fig. S1B). Twenty-two strains showing high recombination by visual inspection only under AID expression were selected for further analysis, whereas those showing high recombination without AID were discarded. After performing fluctuation tests with the 22 selected strains (Fig. S1C), we selected mpl1∆ as the only mutation conferring significant hyperrecombination in the presence of AID.

Table S1.

List of 400 deletion strains that composed the minicollection used for the screening

ORF	Name	ORF	Name	ORF	Name
YAL040C	CLN3	YOR038C	HIR2	YKL032C	IXR1
YAL021C	CCR4	YPL167C	REV3	YKL054C	DEF1
YAL015C	NTG1	YPL164C	MLH3	YKL057C	NUP120
YAL011W	SWC3	YPL139C	UME1	YKL068W	NUP100
YAR002W	NUP60	YPL138C	SPP1	YKL110C	KTI12
YAR003W	SWD1	YPL121C	MEI5	YKL114C	APN1
YLL019C	KNS1	YPL116W	HOS3	YKL149C	DBR1
YLR013W	GAT3	YPL101W	ELP4	YKL160W	ELF1
YLR014C	PPR1	YBR188C	NTC20	YGR044C	RME1
YLR016C	PML1	YBR195C	MSI1	YGR057C	LST7
YLR085C	ARP6	YBR223C	TDP1	YGR066C	—
YLR095C	IOC2	YBR228W	SLX1	YGR067C	—
YLR113W	HOG1	YBR233W	PBP2	YGR102C	GTF1
YML081W	TDA9	YBR245C	ISW1	YGR104C	SRB5
YML062C	MFT1	YDR108W	TRS85	YOR111W	—
YMR153W	NUP53	YDR117C	TMA64	YOR123C	LEO1
YML060W	OGG1	YDR121W	DPB4	YOR144C	ELG1
YML011C	RAD33	YDR146C	SWI5	YOR166C	SWT1
YMR044W	IOC4	YDR359C	EAF1	YOR197W	MCA1
YMR019W	STB4	YDR399W	HPT1	YOR213C	SAS5
YML061C	PIF1	YDR419W	RAD30	YOR228C	MCP1
YMR167W	MLH1	YDR423C	CAD1	YOR246C	ENV9
YMR179W	SPT21	YDR432W	NPL3	YOR258W	HNT3
YMR190C	SGS1	YEL037C	RAD23	YOR288C	MPD1
YMR201C	RAD14	YEL056W	HAT2	YJL206C	—
YMR219W	ESC1	YER032W	FIR1	YJL176C	SWI3
YMR224C	MRE11	YER041W	YEN1	YLR381W	CTF3
YMR284W	YKU70	YER045C	ACA1	YLR385C	SWC7
YNL330C	RPD3	YER051W	JHD1	YLR392C	ART10
YNL309W	STB1	YER068W	MOT2	YLR398C	SKI2
YNL253W	TEX1	YER085C	—	YLR401C	DUS3
YOR023C	AHC1	YGR123C	PPT1	YLR407W	—
YOR032C	HMS1	YGR129W	SYF2	YLR418C	CDC73
YPL178W	CBC2	YGR200C	ELP2	YLR247C	IRC20
YOR051C	ETT1	YGR212W	SLI1	YLR318W	EST2
YOR080W	DIA2	YHL009C	YAP3	YDR163W	CWC15
YOR083W	WHI5	YHR087W	RTC3	YDR169C	STB3
YOR339C	UBC11	YHR124W	NDT80	YDR192C	NUP42
YOR344C	TYE7	YHR134W	WSS1	YDR217C	RAD9
YOR346W	REV1	YHR206W	SKN7	YGL222C	EDC1
YOR386W	PHR1	YCL061C	MRC1	YGL241W	KAP114
YOL001W	PHO80	YCR014C	POL4	YGL244W	RTF1
YOL004W	SIN3	YLR451W	LEU3	YGL251C	HFM1
YOL051W	GAL11	YLR135W	SLX4	YGR001C	EFM5
YPL230W	USV1	YLR154C	RNH203	YGR006W	PRP18
YPL216W	—	YKL009W	MRT4	YPL086C	ELP3
YPL213W	LEA1	YKL020C	SPT23	YPL064C	CWC27
YPL055C	LGE1	YIL079C	AIR1	YJL127C	SPT10
YPL048W	CAM1	YIL084C	SDS3	YJL115W	ASF1
YPL042C	CDK8	YIL008W	URM1	YJL092W	SRS2
YPL037C	EGD1	YFL049W	SWP82	YJL065C	DLS1
YPL015C	HST2	YFL052W	ZNF1	YJL056C	ZAP1
YPL008W	CHL1	YGR270W	YTA7	YJL049W	CHM7
YPL001W	HAT1	YGR275W	RTT102	YJL047C	RTT101
YPR135W	CTF4	YGR288W	MAL13	YER063W	THO1
YPR164W	MMS1	YIR018W	YAP5	YHR041C	SRB2
YPR179C	HDA3	YIR033W	MGA2	YJR078W	BNA2
YPR196W	—	YKL033W-A	—	YJR082C	EAF6
YDR363W-A	SEM1	YKR077W	MSA2	YJR147W	HMS2
YFR034C	PHO4	YKR080W	MTD1	YKR092C	SRP40
YGR249W	MGA1	YMR075W	RCO1	YKR099W	BAS1
YBL006C	LDB7	YMR078C	CTF18	YKR101W	SIR1
YBL008W	HIR1	YMR080C	NAM7	YLR435W	TSR2
YBL019W	APN2	YMR091C	NPL6	YML021C	UNG1
YBL032W	HEK2	YOL090W	MSH2	YNR063W	—
YBL066C	SEF1	YOL100W	PKH2	YJR094C	IME1
YBL088C	TEL1	YOL104C	NDJ1	YJR124C	—
YGL025C	PGD1	YER064C	VHR2	YJR140C	HIR3
YGL013C	PDR1	YER088C	DOT6	YKL005C	BYE1
YPL022W	RAD1	YER092W	IES5	YDL200C	MGT1
YGL043W	DST1	YER095W	RAD51	YDL214C	PRR2
YGL082W	—	YGR134W	CAF130	YDR004W	RAD57
YGL087C	MMS2	YHR191C	CTF8	YBR271W	EFM2
YGL096W	TOS8	YHR193C	EGD2	YBR274W	CHK1
YNL236W	SIN4	YLR032W	RAD5	YCR065W	HCM1
YNL230C	ELA1	YLR035C	MLH2	YCR066W	RAD18
YNL218W	MGS1	YLR052W	IES3	YCR077C	PAT1
YNL156C	NSG2	YMR312W	ELP6	YJR035W	RAD26
YKL213C	DOA1	YNL250W	RAD50	YJR043C	POL32
YKL214C	YRA2	YML095C	RAD10	YJR050W	ISY1
YDR253C	MET32	YML102W	CAC2	YJR060W	CBF1
YDR255C	RMD5	YML103C	NUP188	YNL004W	HRB1
YDR273W	DON1	YML109W	ZDS2	YNL021W	HDA1
YDR279W	RNH202	YML113W	DAT1	YNL025C	SSN8
YDR295C	HDA2	YML121W	GTR1	YNL046W	—
YDR307W	PMT7	YMR106C	YKU80	YNR010W	CSE2
YDR314C	RAD34	YMR125W	STO1	YNR024W	MPP6
YDR317W	HIM1	YPR018W	RLF2	YBL103C	RTG3
YDR334W	SWR1	YPR051W	MAK3	YBR065C	ECM2
YIL017C	VID28	YPR052C	NHP6A	YNL136W	EAF7
YIL024C	—	YPR068C	HOS1	YNL133C	FYV6
YIL040W	APQ12	YPR070W	MED1	YIL128W	MET18
YIL072W	HOP1	YPR101W	SNT309	YIL130W	ASG1
YIR005W	IST3	YJL184W	GON7	YIL149C	MLP2
YIR009W	MSL1	YPR072W	NOT5	YIR002C	MPH1
YIR013C	GAT4	YLR226W	BUR2	YGR056W	RSC1
YNL072W	RNH201	YNL059C	ARP5	YOR141C	ARP8
YNL076W	MKS1	YMR263W	SAP30	YOR162C	YRR1
YNL082W	PMS1	YCR081W	SRB8	YOR161C	PNS1
YNL085W	MKT1	YBR278W	DPB3	YOR172W	YRM1
YNL090W	RHO2	YBR285W	—	YOR191W	ULS1
YNL107W	YAF9	YNL146W	—	YOR208W	PTP2
YNL121C	TOM70	YBR112C	CYC8	YBR033W	EDS1
YDR174W	HMO1	YLL054C	—	YCR084C	TUP1
YGR040W	KSS1	YLR011W	LOT6	YLR399C	BDF1
YGR063C	SPT4	YLR098C	CHA4	YGL240W	DOC1
YKR023W	—	YML080W	DUS1	YGR229C	SMI1
YKR029C	SET3	YMR280C	CAT8	YGR252W	GCN5
YBR131W	CCZ1	YOR001W	RRP6	YAL027W	SAW1
YBR150C	TBS1	YOR006C	TSR3	YNL278W	CAF120
YBR275C	RIF1	YOR295W	UAF30	YOR290C	SNF2
YBR289W	SNF5	YOR297C	TIM18	YOR363C	PIP2
YLR394W	CST9	YOL028C	YAP7	YOR368W	RAD17
YML027W	YOX1	YOL043C	NTG2	YDR066C	RTR2
YML036W	CGI121	YOL068C	HST1	YDR069C	DOA4
YML041C	VPS71	YPL194W	DDC1	YDR075W	PPH3
YMR048W	CSM3	YPL129W	TAF14	YDR092W	UBC13
YMR137C	PSO2	YPL096W	PNG1	YDR099W	BMH2
YIL030C	SSM4	YBR175W	SWD3	YDR369C	XRS2
YOR270C	VPH1	YBR182C	SMP1	YKL113C	RAD27
YOR274W	MOD5	YBR184W	—	YHR034C	PIH1
YOR276W	CAF20	YDR050C	TPI1	YHR079C	IRE1
YOR179C	SYC1	YDR076W	RAD55	YGL058W	RAD6
YKR095W	MLP1	YDR078C	SHU2	YJL103C	GSM1
YLR442C	SIR3	YDR097C	MSH6	YHR204W	MNL1
YNR052C	POP2	YDR123C	INO2	YCL011C	GBP2
YPR023C	EAF3	YDR364C	CDC40	YGR036C	CAX4
YDR443C	SSN2	YDR392W	SPT3	YOL148C	SPT20
YGL151W	NUT1	YDR408C	ADE8	YGR258C	RAD2
YGL173C	XRN1	YDR414C	ERD1	YGL070C	RPB9
YER161C	SPT2	YER035W	EDC2	YDR296W	MHR1
YDL074C	BRE1	YGR159C	NSR1	YGR262C	BUD32
YFL013C	IES1	YGR180C	RNR4	YJL013C	MAD3
YJR047C	ANB1

Fig. S1.

Screening of the selected 400 deletion strains for mutants showing AID-induced hyperrecombination. (A) Cells were transformed with plasmid p313LZGAID, grown in galactose to induce AID expression, and plated on appropriate media to assess the amount of total and recombinant cells, respectively. Wild-type as well as hpr1∆ and mft1∆ THO mutants were used as controls. Representative results obtained with one of the 96-well plate are shown. Positions corresponding to WT, hpr1∆, mft1∆, and mlp1∆ are indicated. (B) For each candidate strain, four independent transformants were grown in either glucose (without AID expression; −AID) or galactose (with AID expression; +AID) and serial dilutions plated on selective media. Strains were assigned to the following categories: (i) strains showing much higher recombination level upon AID expression than without AID expression (Rec. +AID > Rec. −AID) and (ii) strains showing similar recombination levels with or without AID expression (Rec. +AID = Rec. −AID). Representative examples are shown for the different categories. Only strains assigned to the first category (Rec. +AID > Rec. −AID, Right) were selected for further analysis. Worthy of note, the sensitivity of our assay did not allow appreciating AID-dependent recombination increases in strains with high basal recombination level in the absence of AID expression (Left), these strains being consequently assigned to the second category and not retained for further analysis. (C) Recombination analysis of the 22 selected candidates using the L-lacZ direct repeats plasmid-borne system. Wild-type and mft1∆ cells were used as controls. Recombination frequencies were obtained as the median value of six independent colonies. The average and SEM of two independent fluctuation tests are shown for each genotype. Recombination frequency fold-increase obtained for each mutant relative to the wild type in AID expression condition is shown on the right.

Lack of Mlp1/2 Causes R Loop-Dependent Genome Instability and R Loop Accumulation.

Because MLP1 and its paralog MLP2 encode proteins of the nuclear basket with distinct but overlapping functions, we pursued our analysis in mlp1∆, mlp2∆, and mlp1∆ mlp2∆ double mutants in the W303 strain background. First, we showed that AID expression leads to hyperrecombination in mlp1∆, mlp2∆, and mlp1∆ mlp2∆ using the L-lacZ system under the control of the LEU2 promoter (Fig. 1A and Table S2). In the double mutant, the basal recombination frequency is slightly increased compared with the wild type (4.6-fold), whereas the levels of AID-induced recombination are similar to those of either single mutant. Importantly, the AID-dependent increase in recombination was suppressed by RNase H1 overexpression, suggesting that the action of AID is enabled by the presence of R loops in the three strains. Analysis of the transcription dependency of AID-mediated recombination in the L-lacZ system under control of the GAL1 promoter under conditions of low (glucose) or high (galactose) transcription (Fig. 1B, Fig. S2, and Table S2) revealed that hyperrecombination in mlp1/2 mutants depended on both AID expression and transcription, consistent with its dependency on R loops. Recombination was also analyzed in a direct-repeat chromosomal system based on two mutated copies of the HIS3 gene on chromosome XV (38). AID expression substantially increased recombination in an RNase H1-sensitive manner in the three mutants (Fig. 1C and Table S2), consistent with the plasmid-born system results. In this case, recombination frequencies were also slightly increased in the absence of AID expression compared with the wild type, indicating that mutation of Mlp1/2 leads to a weak increase in recombination.

Fig. 1.

Lack of Mlp1/2 leads to AID-dependent genome instability that is suppressed by RNase H1 overexpression. (A) Recombination analysis using the L-lacZ direct-repeat plasmid-borne system under the control of the LEU2 promoter in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ cells that do not express AID (−AID), express AID (+AID), or express both AID and RNase H1 (+AID +RNH1). A scheme of the system is shown (Upper). Average and SEM of independent fluctuation tests are shown (n ≥ 3). Fold increases vs. the wild type with AID are shown on the left. Statistical analyses using a two-tailed unpaired Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. All values are provided in Table S2. (B) Recombination analysis using the L-lacZ system under the control of the GAL1 promoter in cells that do not express AID (−AID) or express AID (+AID) in conditions of low or high transcription. Fold increases with respect to levels under low transcription without AID are shown on the left. Expression levels of the recombination system under high transcription in the different strains were not significantly different (Fig. S2). Details as in A. (C) Recombination analysis using the chromosomal his3-based direct-repeat recombination system. ****P < 0.0001. Details as in A. (D) Percentage of S/G2 cells containing Rad52-YFP foci. Fold increases with respect to the wild type without AID expression are shown on the left. Average and SEM of independent experiments are shown (n ≥ 3). Details as in A. (E) Plasmid loss in cells that do not express RNase H1 (−RNH1) or do express it (+RNH1). Average and SEM of independent experiments are plotted (n ≥ 3). Details as in A.

Table S2.

Values obtained in the recombination, Rad52 foci, and plasmid loss analyses shown in Fig. 1

Strain	Condition	Recombination frequency	% S/G2 cells with Rad52 foci	% Plasmid loss
Fig. 1A
WT	−AID −RNH1	0.84 × 10−3
WT	+AID −RNH1	1.49 × 10−3
WT	+AID +RNH1	0.65 × 10−3
mlp1Δ	−AID −RNH1	0.88 × 10−3
mlp1Δ	+AID −RNH1	9.51 × 10−3
mlp1Δ	+AID +RNH1	1.26 × 10−3
mlp2Δ	−AID −RNH1	0.87 × 10−3
mlp2Δ	+AID −RNH1	8.37 × 10−3
mlp2Δ	+AID +RNH1	1.28 × 10−3
mlp1Δ-mlp2Δ	−AID −RNH1	3.85 × 10−3
mlp1Δ-mlp2Δ	+AID −RNH1	11.60 × 10−3
mlp1Δ-mlp2Δ	+AID +RNH1	3.73 × 10−3
Fig. 1B
WT	−AID low transcription	0.82 × 10−3
WT	−AID high transcription	2.31 × 10−3
WT	+AID low transcription	0.80 × 10−3
WT	+AID high transcription	2.39 × 10−3
mlp1Δ	−AID low transcription	1.27 × 10−3
mlp1Δ	−AID high transcription	2.62 × 10−3
mlp1Δ	+AID low transcription	1.35 × 10−3
mlp1Δ	+AID high transcription	7.28 × 10−3
mlp2Δ	−AID low transcription	1.77 × 10−3
mlp2Δ	−AID high transcription	2.55 × 10−3
mlp2Δ	+AID low transcription	3.10 × 10−3
mlp2Δ	+AID high transcription	9.57 × 10−3
mlp1Δ-mlp2Δ	−AID low transcription	2.17 × 10−3
mlp1Δ-mlp2Δ	−AID high transcription	4.47 × 10−3
mlp1Δ-mlp2Δ	+AID low transcription	1.53 × 10−3
mlp1Δ-mlp2Δ	+AID high transcription	12.65 × 10−3
Fig. 1C
WT	−AID −RNH1	0.47 × 10−4
WT	+AID −RNH1	1.07 × 10−4
WT	+AID +RNH1	0.74 × 10−4
mlp1Δ	−AID −RNH1	0.94 × 10−4
mlp1Δ	+AID −RNH1	4.25 × 10−4
mlp1Δ	+AID +RNH1	1.31 × 10−4
mlp2Δ	−AID −RNH1	0.99 × 10−4
mlp2Δ	+AID −RNH1	4.61 × 10−4
mlp2Δ	+AID +RNH1	1.50 × 10−4
mlp1Δ-mlp2Δ	−AID −RNH1	1.95 × 10−4
mlp1Δ-mlp2Δ	+AID −RNH1	6.07 × 10−4
mlp1Δ-mlp2Δ	+AID +RNH1	1.82 × 10−4
Fig. 1D
WT	−AID −RNH1		2.97
WT	+AID −RNH1		4.68
WT	+AID +RNH1		3.38
mlp1Δ	−AID −RNH1		6.34
mlp1Δ	+AID −RNH1		11.63
mlp1Δ	+AID +RNH1		4.71
mlp2Δ	−AID −RNH1		6.72
mlp2Δ	+AID −RNH1		11.98
mlp2Δ	+AID +RNH1		4.70
mlp1Δ-mlp2Δ	−AID −RNH1		7.88
mlp1Δ-mlp2Δ	+AID −RNH1		13.07
mlp1Δ-mlp2Δ	+AID +RNH1		4.56
Fig. 1E
WT	−RNH1			30.40
WT	+RNH1			40.15
mlp1Δ	−RNH1			74.16
mlp1Δ	+RNH1			41.51
mlp2Δ	−RNH1			65.26
mlp2Δ	+RNH1			69.91
mlp1Δ-mlp2Δ	−RNH1			76.99
mlp1Δ-mlp2Δ	+RNH1			39.41

Fig. S2.

Expression of the L-lacZ direct-repeat plasmid-borne system used in Fig. 1B in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ cells. (A) Scheme of the recombination system. (B) Total RNA was isolated from transformed cells grown in galactose-containing media to midlog phase. Northern blot analysis was performed using a radioactively labeled fragment of the LacZ gene as probe. A probe against the constitutively expressed SCR1 gene was used as loading control. Representative results are shown for each genotype. (C) Quantification of L-lacZ expression as shown in B. LacZ signal was normalized to the SCR1 levels of each sample. Average and SEM of three independent experiments are plotted.

Next, Rad52-foci were monitored as a mark of double-strand breaks (DSBs) by fluorescence microscopy in cells expressing a Rad52–YFP fusion protein. AID expression led to significantly higher percentages of S/G2 cells with Rad52-foci in mlp1∆, mlp2∆, and mlp1∆ mlp2∆ mutants compared with the wild type (Fig. 1D and Table S2). This AID-dependent increase in Rad52 foci was also suppressed by RNase H1 overexpression. The percentage of S/G2 cells with Rad52 foci showed a slight but significant increase in the three mutants strains also in the absence of AID expression, consistent with the idea that mlp1/2 mutants increase genomic instability, in agreement with the implication of the Mlp1/2-bound nucleoporin Nup60 in DSB appearance (39), and as further supported by the analysis of plasmid loss, which increases over twofold in mlp1∆, mlp2∆, and mlp1∆ mlp2∆ compared with the wild type (Fig. 1E and Table S2).

To assess whether Mlp1/2 inactivation increases R loops, DNA–RNA hybrid immunoprecipitation (DRIP) with the S9.6 antibody were done in mlp1∆, mlp2∆, and mlp1∆ mlp2∆ strains at genes previously shown to form DNA–RNA hybrids or to generate transcription-dependent replication defects in R loop-accumulating THO mutants (10, 40, 41). The results show that R loops accumulate at the tested genes (GCN4, PDC1, SPF1, and PDR5) in the three strains (Fig. 2B). RNase H treatment decreased the R loop levels and a very low S9.6 signal was obtained at GAL1 when not transcribed, confirming that the signal was DNA–RNA hybrid specific. In addition, DRIP analysis at the transcribed TRP1 gene located in the intervening sequence of the his3 direct-repeat recombination system (Fig. 1C) showed also R loop accumulation in three mutants (Fig. 2C), consistent with the results of the recombination assays. Altogether, these results indicate that the lack of Mlp1/2 increases cotranscriptional R loops that may potentially cause genome instability. Because similar results were obtained in single and double mutants, we hereafter focus on mlp1∆ strains.

Fig. 2.

R loops accumulate at transcribed genes in mlp1/2 mutants. (A) Schematic view of the analyzed genes and amplicons. (B) DRIP using the S9.6 antibody in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ asynchronously growing cells. Where indicated, samples were treated with RNase H (RNH). S9.6 signal were normalized to the wild-type value in each experiment. Average and SEM of independent experiments are shown (n ≥ 3). Statistical analyses as in Fig. 1A. *P < 0.05; **P < 0.01; ***P < 0.001. (C) DRIP in the his3-based direct-repeat recombination system. The S9.6 signal at the GNC4 and GAL1 genes were used as controls. Details as in B.

R Loop Accumulation Interferes with Replication in mlp1∆ Cells.

Interference with the replication process has emerged as one of the major causes of R loop-mediated genome instability (42, 43). Consequently, we assayed replication fork (RF) progression in mlp1∆ cells by different means. First, we analyzed replication by FACS and pulse-field gel electrophoresis (PFGE) in mlp1∆ cells (Fig. S3). No significant differences could be appreciated between the mlp1∆ and wild-type strains, independently of AID expression or the presence of hydroxyurea (HU), which slows down replication. Because this method might not be sufficiently sensitive, we analyzed replication by monitoring BrdU incorporation in cells upon release from α-factor–mediated G1 arrest in the vicinity of the early firing origins ARS508 and ARS1211. These origins were chosen because they are situated in close vicinity to the SPF1 and PDC1 genes, respectively, in which R loops accumulate in mlp1/2 mutants (Fig. 2B) and in head-on orientation in respect to replication. DNA was immunoprecipitated with anti-BrdU antibody and subjected to real-time qPCR at the 5′ and 3′end of the transcription units and the ARS sequences. mlp1∆ cells showed mild defects in replication compared with wild-type cells (Fig. 3A and Fig. S4), which are also seen in the absence of HU (Fig. S5). Importantly, the difference in BrdU incorporation observed between wild-type and mlp1∆ cells was completely lost in cells overexpressing RNase H1 at all analyzed regions, indicating that R loop accumulation is likely responsible for the replication delay (Fig. 3A and Fig. S4). To appraise whether the replication defects might be due to the head-on orientation of replication–transcription of the systems tested, we analyzed BrdU incorporation at ARS1021, an early firing ARS located next to the codirectionally orientated gene ECM17 (Fig. 3B and Fig. S4). RNase H1-sensitive replication defects were observed at this locus as well, supporting the conclusion that R loop accumulation leads to general replication defects in mlp1∆ cells.

Fig. S3.

Cell-cycle progression analyses of mlp1∆ cells. (A) Flow cytometry analyses (FACS) of mlp1∆ and wild-type cells during release from α-factor–mediated G1 arrest in normal condition (Top), in the presence of 20 mM hydroxyurea (+HU; Middle), and in cells expressing human AID (+AID; Bottom). FACS analyses were performed with a FACSCalibur (BD Bioscience) using CellQuest software. (B) Chromosome XII species revealed by hybridization with ADE5 in pulse-field electrophoresis (PFGE) of DNA from mlp1∆ and wild-type cells released from α-factor–mediated G1 arrest in normal condition (Upper) and in the presence of 20 mM hydroxyurea (+HU; Lower). Nonlinear chromosomes (NLCs), which include replication intermediates, correspond to the signal coming from the gel well. The NLC signal was quantified with respect to the total signal of each lane. FLC, full-length linear chromosomes. Representative results and quantification from three independent experiments are shown. Average and SEM are plotted. PFGE was performed using standard protocol.

Fig. 3.

Replication progression is impaired in mlp1∆ cells. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and amplicons are depicted (Top). Experiments were done in cells treated with 20 mM hydroxyurea (HU) not overexpressing RNase H1 (−RNH1) or overexpressing RNase H1 (+RNH1). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average and SEM of independent experiments are shown (n = 3). The P values calculated by the Wilcoxon signed-rank test are shown for each condition. Plotted values are normalized to the average signal obtained for the wild type at the 25-min time-point in the +510 region for each experiment. The graphs obtained without normalization to the wild-type value are shown in Fig. S4. (B) BrdU incorporation upon release of G1-arrested cells at ARS1021. Signal in the +625 region was used for normalization. Details as in A.

Fig. S4.

Representation of the BrdU incorporation analyses shown in Fig. 3 and at ARS1211 without normalization with wild-type values. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and localization of the amplified regions are depicted (Top). The analysis was performed in cells treated with 20 mM hydroxyurea (HU) that did not overexpress RNase H1 (−RNH1) or did overexpress RNase H1 (+RNH1). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average from three independent experiments and corresponding SEM are shown. The P values calculated by the Wilcoxon signed-rank test are shown for each condition. (B) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1211. Other details as in A. (C) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1021. Other details as in A.

Fig. S5.

Replication progression is impaired in mlp1∆ cells. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and localization of the amplified regions are depicted (Top). The analysis was performed in untreated cultures (−HU) or in cultures treated with 20 mM hydroxyurea (+HU). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average from three independent experiments and corresponding SEM are shown. The P values calculated by the Wilcoxon signed-rank test are shown for each region. (B) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1211. Other details as in A.

Gene Tethering to the NPC Prevents R Loop Accumulation.

Recent work has shown that Mlp1 is a target for intra-S checkpoint kinase Rad53, and that Mlp1 phosphorylation results in the release of DNA gated at the NPC (37). So, we took advantage of reported mutants carrying a phosphomimetic or a nonphosphorylatable mlp1 allele (mlp1-S1710D and mlp1-S1710A, respectively) (37) and analyzed AID-mediated recombination and Rad52-foci (Fig. 4 A and B and Table S3). The results are consistent with our hypothesis that gene gating may prevent the formation of R loops and their associated genomic instability because the mlp1-S1710D mutant that mimics constitutive checkpoint activation—that in turn supposes release of the damaged DNA from the NPC—behaved similarly to the gene gating-deficient mlp1∆ strain in both assays. On the contrary, the mlp1-S1710A mutant that does not allow checkpoint-dependent release from the NPC did not appear to show R loop-mediated increase in recombination or Rad52 foci accumulation. Consistently, plasmid loss was also exacerbated in the D allele compared with the A allele (Fig. 4C and Table S3). Direct assessment by DRIP with the S9.6 antibody confirmed R loop accumulation in the D mutant (Fig. 4D). In the A mutant, the S9.6 signal was also increased, but it was RNase H-resistant, indicating that it does not correspond to R loops.

Fig. 4.

R loop-dependent genome instability in mlp1 mutants mimicking constitutive intra-S checkpoint activation or blind to checkpoint activation. (A) Recombination analysis using the L-lacZ system under the control of the LEU2 promoter in mlp1S1710D (mlp1D) and mlp1S1710A (mlp1A) cells that do not express AID (−AID), express AID (+AID), or express both AID and RNase H1 (+AID +RNH1). *P < 0.05; **P < 0.01. Details as in Fig. 1. (B) Percentage of S/G2 cells containing Rad52-YFP foci. Details as in A. (C) Plasmid loss in cells that do not express RNase H1 (−RNH1) or do express RNase H1 (+RNH1). ***P < 0.001. Details as in A. (D) DRIP using the S9.6 antibody. Where indicated, samples were pretreated with RNase H (RNH). *P < 0.05; **P < 0.01; ***P < 0.001. Details as in Fig. 2. Experiments of B–D were performed concomitantly with those shown in Figs. 1 and 2, so values for wild-type cells are the same.

Table S3.

Values obtained in the recombination, Rad52 foci, and plasmid loss analyses shown in Fig. 4

Strain	Condition	Recombination frequency	% S/G2 cells with Rad52 foci	% Plasmid loss
Fig. 4A
WT	−AID −RNH1	1.28 × 10−3
WT	+AID −RNH1	1.52 × 10−3
WT	+AID +RNH1	1.64 × 10−3
mlp1D	−AID −RNH1	0.93 × 10−3
mlp1D	+AID −RNH1	6.92 × 10−3
mlp1D	+AID +RNH1	1.19 × 10−3
mlp1A	−AID −RNH1	0.70 × 10−3
mlp1A	+AID −RNH1	0.60 × 10−3
mlp1A	+AID +RNH1	1.19 × 10−3
Fig. 4B
WT	−AID −RNH1		2.97
WT	+AID −RNH1		4.68
WT	+AID +RNH1		3.38
mlp1D	−AID −RNH1		5.12
mlp1D	+AID −RNH1		7.63
mlp1D	+AID +RNH1		4.13
mlp1A	−AID −RNH1		3.51
mlp1A	+AID −RNH1		3.24
mlp1A	+AID +RNH1		3.80
Fig. 4C
WT	−RNH1			30.40
WT	+RNH1			40.15
mlp1D	−RNH1			75.76
mlp1D	+RNH1			84.25
mlp1A	−RNH1			49.84
mlp1A	+RNH1			48.96

Next, we asked whether R loops in mlp1∆ cells could be suppressed by artificially tethering the DNA to the NPC. For this we used the reported artificial anchoring system of the GAL1 locus to the NPC (44). Artificial tethering is achieved through the insertion of LexA binding sites downstream of GAL1 and expression of a LexA–Nup60 fusion protein containing a nuclear localization signal (NLS). An NLS-containing LexA is used as a control with no NPC anchoring. Samples were collected at different time points upon transcriptional activation of the locus in galactose medium. Transcription was followed by northern of GAL1 and ChIP of RNA polymerase II (RNAP II) occupancy, and R loops where detected by DRIP (Fig. 5 B–E). In the control experiment in which the GAL1 locus was not tethered to the NPC, transcriptional activation occurred less efficiently in mlp1∆ than in the wild type, and R loops were detected from the first time-point after transcriptional activation, in agreement with the idea that R loops are concomitant with transcription. When the LexA–Nup60 fusion protein was expressed in mlp1∆ cells, transcription activation was improved and R loops did not accumulate. These results indicate that artificial tethering of the GAL1 gene abolishes both the transcriptional defects and the accumulation of R loops at this locus in mlp1∆ cells. This role of gene gating in preventing R loops is not specific to cells lacking the nuclear basket Mlp1 protein, because analysis of R loops with and without NPC tethering in mlp1∆ and THO-deficient hpr1∆ cells revealed that artificial anchoring of the GAL1 locus to the NPC did suppress R loop formation in both mlp1∆ and hpr1∆ cells (Fig. 5F). LexA–Nup60 expression per se did not have a global effect on R loop formation, as shown by analysis of the unrelated and R loop-rich rDNA locus that showed equal R loop levels in wild-type, mlp1∆, and hpr1∆ cells expressing LexA and LexA–Nup60 at the rDNA (Fig. S6). These results demonstrate that gene gating plays a determinant role in the prevention of R loop accumulation in yeast.

Fig. 5.

Artificial tethering to the nuclear pore is sufficient to suppress cotranscriptional R loops in mlp1∆ and hpr1∆ cells. (A) Scheme of the artificial tethering system. Binding of a LexA–Nup60 fusion protein to LexA binding sites (BS) inserted downstream of the GAL1 gene anchors the GAL1 to the nuclear pore. (B) Time-course analysis of GAL1 mRNA upon transcription induction in wild-type and mlp1∆ cells expressing either LexA or LexA–Nup60. Representative results are shown. (C) Quantification of GAL1 mRNA induction as shown in B. GAL1 signal was normalized to the SCR1 levels of each sample. Average and SEM of independent experiments are plotted (n ≥ 3). The P values were calculated by the Wilcoxon signed-rank test. (D) Time-course ChIP of the RNAP II large subunit Rpb1. In each experiment, ChIP values were normalized to the 0 time-point of the wild type. Location of the amplicons relative to GAL1 gene is indicated (Upper). Details as in C. (E) Time-course DRIP analysis with the S9.6 antibody. DRIP values were normalized to the 0-min time-point in each strain. Details as in D. (F) DRIP using the S9.6 antibody in wild-type, mlp1∆, and hpr1∆ cells grown to midlog phase in galactose medium and expressing either LexA or LexA–Nup60. *P < 0.05; **P < 0.01. Details as in Fig. 2. DRIP data of the rDNA locus is shown in Fig. S6. (G) Model for R loop formation in the nucleoplasm. Gating of a transcribed locus to the NPC prevents formation of R loops. This likely occurs thanks to the prompt export of the nascent mRNP that minimizes the probability of back-hybridization to the template DNA. In gene gating defective mutants such as mlp1∆, transcribed genes remain in the nucleoplasm and R loops accumulate, leading to moderate genome stability.

Fig. S6.

DNA–RNA immunoprecipitation using the S9.6 antibody in wild-type, mlp1∆, and hpr1∆ cells grown in galactose and expressing either LexA or LexA–NUP60 fusion protein at the rDNA locus. Where indicated, samples were treated with RNase H (RNH) before immunoprecipitation. Average of signal values of R loop detection obtained from at least three independent experiments and the corresponding SEM are shown. Statistical analyses were performed with a two-tailed unpaired Student t test. *P < 0.05; **P < 0.01.

Discussion

R loops cause genome instability and have been linked with a number of neurodegenerative diseases and with tumorigenesis (42, 45, 46). Using AID expression as a tool, we identified MLP1 in a screen for genes involved in preventing R loop accumulation in budding yeast. Our analyses of mlp1, mlp2, and mlp1 mlp2 mutants revealed that R loops accumulate at transcribed genes in either of these strains, leading to increased genome instability as seen by AID-dependent hyperrecombination, increased Rad52 foci, and plasmid loss. In agreement with the idea that interference with the replication process is one of the major causes of R loop-mediated genome stability (42, 43), deletion of MLP1 causes mild replication defects that are suppressed by RNase H1 overexpression. Such replication defects were observed without AID expression, consistent with the moderate increase in recombination, Rad52 foci, and plasmid loss observed in untreated mlp1/2 mutants, suggesting that R loops accumulated in the absence of the Mlp1/2 moderately challenge genome stability.

In yeast, Mlp1 associates with highly transcribed genes and is required for gene gating at the NPC (35, 36). Our results demonstrate that tethering of the GAL locus to the NPC is sufficient to suppress R loops at this locus in mlp1 cells, as well as in the hpr1 THO mutant, suggesting that gene gating does generally prevent R loop formation. Formation of export-competent mRNPs is important in preventing R loops and their accompanying genome instability (2), whereas gene gating facilitates mRNA export (34). Covering of the nascent RNA with mRNP biogenesis factors and physical transport through nuclear pores would prevent the nascent RNA from forming a DNA–RNA hybrid. In support of this view, overexpression of RNA binding factors such as Tho1, Nab2, or Sub2/UAP56 suppresses the transcription-dependent hyperrecombination of THO mutants (5, 47, 48). Noteworthy, Nab2 physically interacts with Mlp1 and is believed to be required for proper docking of the mRNP to the Mlp1/2 platform (31, 32). Consistently, mlp1/2 or nab2 mutations lead to frequent release of the mRNP into the nucleoplasm (49). Because the THSC/TREX-2 mRNP biogenesis complex is required for gene gating (50), it would be interesting to investigate whether Nab2 overexpression restores gene gating in THSC/TREX-2 mutants.

Our results show that mlp1 and hpr1 cells share a similar behavior with respect to their R loops being suppressed by artificial tethering of a chromosomal locus, whereas their impact on genome stability is far apart, hpr1 being extremely hyperrecombinant in a transcription-dependent manner (51) and mlp1 showing moderately increased transcription-dependent recombination only after AID expression (Fig. 1). These differences might rely on the fact that, unlike Mlp1, THO is recruited to active genes by the elongating RNAP II and participate in transcription (10, 52, 53). In addition, altered chromatin structure has been associated with R loops in THO mutants both in Caenorhabditis elegans and yeast (54), which could represent a serious threat to RF progression. It is thus possible that THO mutants accumulate genome-threatening situations, which may interfere with the cellular processes that normally deal with pathological R loops or stalled forks, whereas these situations are not produced in mlp1 cells. However, according to the recent evidence that a second step of R loop stabilization is required for R loop-mediated genome instability, as would occur in hpr1 mutants but not in specific histone H3 and H4 mutants (23), it is also possible that R loops in mlp1/2 mutants might not be efficiently stabilized.

Altogether, our data supports a model in which physical proximity to the NPC would protect transcribed genes from R loop accumulation (Fig. 5G). In NPC-gated genes, the nascent mRNP would readily get exported out of the nucleus, reducing the probability of back-hybridization with the DNA. In addition, a more efficient mRNP assembly or the NPC itself may contribute to prevent R loop formation at gated genes. Consequently, R loop accumulation would occur in DNA transcribed in the nucleoplasm, as seen in mlp1/2 gene-gating defective cells. This model is consistent with the facts that both transcription elongation and RNA export are impaired in THO mutants (5, 55) and that R loops in THO- and Mlp1-depleted cells are suppressed by artificial DNA tethering to the NPC (Fig. 5F).

Finally, our study suggests that replication stress-induced release of transcribed genes from nuclear pores (37) would lead to R loop formation in the nucleoplasm, which may serve to further amplify the checkpoint activation cascade and signal the sites of RF stalling. Consistent with this idea, different studies suggest that R loops may accumulate in cells undergoing replicative stress (20, 25, 27) and participate in DNA damage signaling to activate ATM in human cells (56). Interestingly, the NPC-associated helicase DDX19 relocates to the core of the nucleus in response to replication stress or DNA damage to remove R loops (20). Our study thus opens new perspectives to understand the role of RNA in genome dynamics and the control of genome integrity as a function of nuclear location and raises the question of whether the human Mlp1/2 homolog TPR may fulfill the same functions in replication checkpoint and R loop prevention as in yeast.

Materials and Methods

DRIP Assays.

Genomic DNA was carefully extracted and enzymatically digested. DNA–RNA hybrids were immunoprecipitated with the S9.6 antibody, with or without RNase H pretreatment. S9.6 signals were determined by dividing the immunoprecipitated S9.6 signal between the input for each sample as quantified by real-time-qPCR. See SI Materials and Methods for details.

Replication Analysis.

BrdU ChIPs were performed in cells released from G1 arrest in the presence of 200 µg/mL BrdU. Immunoprecipitation was performed using monoclonal anti-BrdU antibody. See SI Materials and Methods for details.

Time-Course Analysis of GAL1 Induction.

Cells were grown at 30 °C to midlog phase in SC medium with 2% raffinose. A total of 2% galactose was added to the medium and samples taken at the indicated time. Isolation and Northern analysis of GAL1 and SCR1 mRNAs were performed following standard procedures. RNAP II ChIP was performed with the monoclonal anti-Rpb1 antibody (8WG16; Covance) and signals were quantified by real-time-qPCR. See SI Materials and Methods for details.

Miscellanea.

Yeast strains, plasmids, and primers used are listed in Tables S4 and S5. Recombination and plasmid loss assays, Rad52-YPF foci, FACS, and PFGE were performed using standard procedures. See SI Materials and Methods for details.

Table S4.

Yeast strains and plasmids used in this study

Strain or plasmid	Relevant genotype or feature	Source
SYRB1-4C	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX ura3::URA3/GPD-TK (7x)	This study
yFGB01	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX ura3::URA3/GPD-TK (7x) mlp1Δ::KanMX	This study
yFGB02	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX ura3::URA3/GPD-TK (7x) mlp2Δ::KanMX	This study
yFGB03-1a	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX ura3::URA3/GPD-TK (7x) mlp1Δ::KanMX mlp2Δ::KanMX	This study
yFGB04	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX	This study
yFGB05	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX mlp1Δ::KanMX	This study
yFGB06	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX mlp2Δ::KanMX	This study
yFGB07	MATa ura3-1 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 RAD5 bar1Δ::HygMX mlp1Δ::KanMX mlp2Δ::KanMX	This study
CY11555	MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ADE2 CAN1 RAD5 mlp1S1710A	(37)
CY11531	MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ADE2 CAN1 RAD5 mlp1S1710D	(37)
FSY5216	MATa ura3 ade2 his3 leu2 trp1 Nup49-GFP LacI-GFP-HIS3 LexA BS LacO at GAL10 TRP	(44)
yFGB07-1a	MATα ura3 ade2 his3 leu2 trp1 Nup49-GFP LacI-GFP-HIS3 LexA BS LacO at GAL10 TRP	This study
yFGB07-1b	MATα ura3 ade2 his3 leu2 trp1 Nup49-GFP LacI-GFP-HIS3 LexA BS LacO at GAL10 TRP mlp1Δ::KanMX	This study
yFGB08	MATα ura3 ade2 his3 leu2 trp1 Nup49-GFP LacI-GFP-HIS3 LexA BS LacO at GAL10 TRP hpr1Δ::HIS3	This study
344115B leu+	Matα his3-513::TRP1::his3-537 ura3-52 trp1	(23)
yFGB09	Mata his3-513::TRP1::his3-537 ura3 trp1 bar1Δ::HygMX	This study
yFGB10	Mata his3-513::TRP1::his3-537 ura3 trp1 bar1Δ::HygMX mlp1Δ::KanMX	This study
yFGB11	Mata his3-513::TRP1::his3-537 ura3 trp1 bar1Δ::HygMX mlp2Δ::KanMX	This study
yFGB12	Mata his3-513::TRP1::his3-537 ura3 trp1 mlp1Δ::KanMX mlp2Δ::KanMX	This study
pLZGAID	YCp containing the pL-lacZ system and the human AID gene under the GAL1 promoter	(23)
pRS313	YCp vector based on the HIS3 marker	(60)
p313LZGAID	YCp containing the pL-lacZ system and the human AID gene under the GAL1 promoter	This study
pRS313-GALRNH1	YCp containing the RNH1 gene under the GAL1 promoter	This study
pRS314GLlacZ	YCp containing the L-lacZ system under the GAL1 promoter	(61)
pCM184	YCp plasmid containing the tetO promoter and TRP1 marker	(62)
pCM189	YCp plasmid containing the tetO promoter and URA3 marker	(62)
pCM184AID	YCp containing the AID ORF cloned from pRS316GALAID (29) into pCM184 NotI site	This study
pCM189AID	YCp containing AID ORF under the tetO promoter	(63)
pCM184RNH1	YCp containing RNH1 ORF under the tetO promoter	(63)
pWJ1213	YCp containing the RAD52-YFP fusion	(64)
p414GAL	YCp containing the GAL1 promoter	(65)
pRS414-GALAID	YCp containing the human AID ORF under the GAL1 promoter	(29)
pRS315	YCp vector based on the LEU2 marker	(60)
pRS315-GALRNH1	YCp carrying the RNH1 ORF under the GAL1 promoter	(10)
pBTM116-URAr-LexA	Yeast vector containing LexA DNA-binding ORF	(44)
pBTM116-URAr-LexA-Nup60	Yeast vector containing NUP60 ORF fused to LexA DNA-binding ORF	(44)

Table S5.

Primers used in this study

Primers	Sequence
MLP1 A	CTGATAGATATATTGCTGCC
MLP1 B	AACATTCAAAACACAAACCG
MLP2 A	AAGAAGAAAACAATATCGGCG
MLP2 B	ATACTTAACTACTAGTACGG
GCN4 3′ A	TTGTGCCCGAATCCAGTGA
GCN4 3′ B	TGGCGGCTTCAGTGTTTCTA
PDC1 A	CCTTGATACGAGCGTAACCATCA
PDC1 B	GAAGGTATGAGATGGGCTGGTAA
PDR5 A	TACGTCTTGTTTCGGCCTTAATC
PDR5 B	GTCAGAGGCTATATTTCACTGGAGAA
ARS508 A	AGATTCTTTGAACACGGTCTGTCA
ARS508 B	TGTGCTAAACCACTCAGTTGGAA
ARS508 +3508 A	CCCGTGGTAAACCTTTAGAAA
ARS508 +3508 B	ATATGAACGGCAAATTGAGAC
ARS508 +510 A	AGTCATTAATAGCAAAGCCGT
ARS508 +510 B	GGTCCTTTGATGTAACGATCA
ARS1211 A	CGGCTTACCGGTCTTGAAAAT
ARS1211 B	GGAATACTTTTGCTTGAGTTGTTTAGTTT
ARS1211 +125 A	GTTTCCTCCACCTCCTTTGTGT
ARS1211 +125 B	TGACCGATATATTGTGTTTCTATACTGTGT
ARS1211 +2505 A	CGTTCAATTCGTTGGCGTTAC
ARS1211 +2505 B	TTAACACCGTTTTCGGTTTGC
ARS1021 A	CCCATTTCGGCGGCTAAT
ARS1021 B	TAGAAGCCATTGATGGTATTGTACATT
ARS1021 +4597 A	GGTTGCCCTAACGGTTGTTC
ARS1021 +4597 B	TGGAGCTTTACCAACAAGAGCTAA
ARS1021 +625 A	TGCTCCCCAAAATAAAGTGTTCTAC
ARS1021 +625 B	AGCCCTTTGAAGGATGAATGAC
Chr. V A	TGCCTGCACGCCATTGT
Chr. V B	TTCCCCACGGAAAGTTGTATCT
SCR1 A	GTTCAGGACACACTCCATCC
SCR1 B	AGGCTGTAATGGCTTTCTGG
GAL1 A	ACGAGTCTCAAGCTTCTTGC
GAL1 B	TATAGACAGCTGCCCAATGC
LacZ400 Fwd	TGTACGGTACCATGGCGATTACCGTTGATG
LacZ400 Rev	TAGTAGGATCCTTATTTTTGACACCAGACC
GAL1 3′ A	AAAGAAGCCCTTGCCAATGA
GAL1 3′ B	CATTTTCTAGCTCAGCATCAGTGATC
GAL1 5′ A	TGAGTTCAATTCTAGCGCAAAGG
GAL1 5′ B	TTCTTAATTATGCTCGGGCACTT
INT A	TGTTCCTTTAAGAGGTGATGGTGAT
INT B	GTGCGCAGTACTTGTGAAAACC
TRP1 A	CGTCCAACTGCATGGAGATG
TRP1 B	TGGCAAACCGAGGAACTCTT
rDNA 18S A	TCAACTTTCGATGGTAGGAT
rDNA 18S B	GGAATCGAACCCTTATTCCC

SI Materials and Methods

Yeast Strains, Plasmids, and Primers.

Yeast strains used for the screening are listed in Table S1. All other strains used in this work were isogonics to W303, and are listed in Table S4. mlp mutants were generated by replacement of the MLP1 or MLP2 genes with the KanMX cassette in strain SYRB1-4C. The URA3/GPD-Tk (7×) sequence integrated at the ura3 locus was popped out by growing the strains in 5-fluoroorotic acid-containing synthetic complete plates without uracil to generate yFGB04, yFGB05, and yFGB06 strains. Strains baring the chromosomal his3-based recombination system were obtained by genetic crosses with strain 344-15B-Leu+ (23). Strains baring the artificial anchoring construction were obtained by genetic crosses with strains FSY5216 and FSY5217 (44). Plasmids and primers used in this work are listed in Tables S4 and S5. Plasmid p313LZGAID was generated by cloning the HIS3-containing ApaI–DraIII fragment from pRS313 into ApaI–DraIII-digested pLZGAID (23).

Large-Scale Yeast Transformation.

Large-scale transformation was performed as previously described (23). Briefly, cells were grown in 2× YPAD for 2 d at 30 °C and washed in 0.1 M LiAc 10 mM TE before transformation. Cells were resuspended and incubated 30 min at 30 °C and 20 min at 42 °C in the transformation mix (500 ng plasmid DNA, 30% PEG, 100 mM LiAc, 1× TE, 80 µg/mL salmon sperm DNA). Transformed cells were washed and grown in appropriate selective medium.

Recombination and Plasmid-Loss Assays.

Recombination frequencies were determined as the average value of the median frequencies obtained from at least three independent fluctuation tests with the indicated recombination systems. Each fluctuation test was performed from six independent colonies according to standard procedures (57).

Percentage of plasmid loss was determined as the average value of the median percentages of cells that lost centromeric plasmid pRS315 upon growth in nonselective media obtained from at least three independent fluctuation tests. Each test was performed with six independent colonies.

Detection of Rad52-YFP Foci.

Rad52-YFP foci were visualized in cells transformed with plasmid pWJ1213 with a DM600B microscope (Leica) as previously described (58) with minor modifications. Individual transformants were grown to early log-phase, fixed for 10 min in 0.1 M K_iPO₄ pH 6.4 containing 2.5% formaldehyde, washed twice in 0.1 M K_iPO₄ pH 6.6, and resuspended in 0.1 M K_iPO₄ pH 7.4. At least 200 S/G2 cells were analyzed for each transformant. Average values obtained from at least three independent transformants are plotted for each genotype.

DRIP Assays.

DNA–RNA immunoprecipitation was performed as previously described (40). Briefly, DNA was carefully extracted from spheroplasts with chloroform:isoamylalcohol (24:1) followed by isopropanol precipitation. Precipitated DNA was spooled on a glass rod, washed twice with 70% EtOH, gently resuspended in TE, and enzymatically digested with HindIII, EcoRI, BsrGI, XbaI, and SspI. Samples were split and treated with Escherichia coli RNase H (NEB) or mock treated. DNA–RNA hybrids immunoprecipitation was performed by overnight incubation with Protein A Dynabeads (Invitrogen) coated with the S9.6 antibody (hybridoma cell line HB-8730) at 4 °C. DNA was treated with proteinase K and purified with the MACHEREY-NAGEL DNA kit. Real-time qPCR was performed at the indicated regions. S9.6 signal was determined by dividing the immunoprecipitated signal to the input for each sample. The relative abundance of DNA–RNA hybrid immunoprecipitated in each region was normalized to the signal of the WT signal without RNase H treatment.

Replication Analysis.

Cells were grown in SC medium, incubated for 2.5 h with 0.125 μg/mL α-factor (Biomedal), washed twice in prewarmed SC medium, and released from G1 arrest in the presence of 200 μg/mL BrdU (Sigma) by addition of 1 μg/mL pronase. Where indicated, release was performed in the presence 20 mM hydroxyurea. A total of 0.1% sodium azide was added to each sample, and cells were broken in a multivortex at 4 °C in lysis buffer (50 mM Hepes–KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and sonicated. Immunoprecipitation was performed using Protein A Dynabeads (Invitrogen) coated with monoclonal anti-BrdU antibody (MBL). Input and immunoprecipitated DNA were analyzed by real-time qPCR. Relative BrdU incorporation at a given region was calculated relative to the signal at a late replicating region (chromosome V, position 242210–242280; ref. 10) in the same sample.

Time-Course Analysis of GAL1 Induction.

Cells were grown at 30 °C to midlog phase in SC medium with 2% raffinose. Galactose was added to the medium (2% final concentration) and samples taken at the indicated time. RNA isolation and Northern analysis with GAL1 and SCR1 probes were performed following standard procedures. RNAP II chromatin immunoprecipitation was performed as previously described (59). A monoclonal anti-Rpb1 antibody (8WG16; Covance) was used, and real-time qPCR performed at the indicated regions. DRIP assay was performed as indicated previously.