Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells

Abstract

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N⁶-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

Introduction

Biochemical and structural studies with translesion synthesis (TLS)² DNA polymerases (Pols) have indicated a high degree of specificity in the types of DNA lesions they can replicate through (1). Thus, the ability to accommodate two template residues in its active site provides Polη the proficiency for replicating through the covalently linked cyclobutane pyrimidine dimer (CPD) (2–6). The adoption of a syn conformation by the purine template in the Polι active site for forming a Hoogsteen base pair with the incoming nucleotide enables it to insert nucleotides opposite DNA adducts which impair Watson–Crick (W-C) base pairing or impinge upon the DNA minor groove (7–10). TLS studies in human cells have corroborated the roles and mechanisms inferred from biochemical and structural studies of Polη, Polι, and other TLS Pols (11–15).

In vitro studies of purified Polθ, an A family Pol, have suggested that in contrast to Polη or Polι, it lacks the specificity for replicating through DNA lesions; and compared with TLS mediated by Pols with high specificity, Polθ acts in a more error-prone manner (12, 13). In human cells, for example, Polθ functions in TLS opposite two very different types of lesions, CPDs and 1,N⁶-ethenodeoxyadenosine (ϵdA). TLS opposite CPDs occurs either by a Polη-dependent error-free pathway or by an alternative error-prone pathway in which Polθ inserts a nucleotide opposite the 3′ pyrimidine residue of a CPD from which Polκ or Polζ subsequently extend synthesis (13). TLS opposite the ϵdA adduct, which is generated from interaction of DNA with aldehydes derived from lipid peroxidation (16, 17), and which impairs W-C base pairing, operates via two major pathways dependent upon Polι/Polζ and Polθ, respectively, in which the sequential action of Polι and Polζ promotes error-free TLS and Polθ performs error-prone TLS (12). A third pathway dependent upon Rev1 polymerase activity makes a relatively minor contribution (12). Apart from these Pols, no other Pols such as Polη, Polκ (12), or Polν are required for TLS opposite this adduct in human cells.

The ability of Polι to insert nucleotides opposite the ϵdA adduct by Hoogsteen base pairing and the proficiency of Polζ for extending synthesis from the nucleotide opposite ϵdA explains the roles these Pols play in TLS through ϵdA in human cells (9, 12). Because Polθ replicates DNA by utilizing classical W-C base pairing, ϵdA would present a strong block unless the adduct adopts an extrahelical position in the Polθ active site; hence, Polθ replicates through ϵdA using a mechanism similar to the one it uses for TLS through an abasic (AP) site. The observation that purified Polθ replicates through both the ϵdA and AP lesions by inserting an A is consistent with ϵdA adopting an “AP” mode in the Polθ active site (12). However, in striking contrast to the extremely error-prone TLS opposite ϵdA by purified Polθ, Polθ-dependent TLS in human cells operates in a predominantly error-free manner wherein Polθ incorporates over 90% T opposite ϵdA (12). Such error-free TLS could occur in human cells only if the ϵdA adduct adopts a syn confirmation in the Polθ active site and forms a Hoogsteen base pair with the T residue.

To test the validity of the hypothesis that Polθ adopts a different mechanism for TLS in human cells than in purified Polθ, in this study, we analyze the effects of mutations in the two highly conserved tyrosine residues in the Polθ active site on TLS opposite ϵdA by purified Polθ and on TLS in human and mouse cells. Our findings that these mutations affect TLS by purified Polθ in a dramatically different way than they affect TLS in human and mouse cells strongly support the premise that the Polθ active site is configured differently for TLS in human cells than in purified Polθ.

Results

Conserved tyrosine residues in Polθ fingers domain

The O-helix in the fingers domain is conserved among A-family Pols. Within the O-helix, the Tyr-2391 residue in human Polθ is conserved among all of the eukaryotic, prokaryotic, and phage A-family DNA polymerases, whereas Tyr-2387 in human Polθ is conserved in both of the eukaryotic A-family Pols Polθ and Polν, but it is not conserved in Escherichia coli PolI, Taq polymerase, or T5 Pol (Fig. 1). The ternary crystal structures of human Polθ have revealed that the Tyr-2387 residue contacts the β-phosphate of the incoming nucleotide, and Tyr-2391 lies beneath the template residue (18).

Figure 1.

Homology among human Polθ and other A-family DNA polymerases. The region encompassing the Polθ O-helix within the finger domain is shown. The arrows above the alignment indicate helical elements as determined from crystallography (Protein Data Bank entry 4X0Q). The amino acid positions in each protein are indicated on the right and left. Mutations in the Tyr-2387 and Tyr-2391 residues (in boldface type) of human Polθ were examined in this study. Homologous residues are indicated below, where an asterisk indicates identical residues, and periods and colons indicate moderately and highly homologous residues, respectively. Hs, human Polθ; Mm, mouse Polθ; Bt, bovine Polθ; Dm, Drosophila melanogaster Mus308; Ec, E. coli PolI, Taq, Thermus aquaticus PolI.

Indispensability of Tyr-2387 and Tyr-2391 for TLS through ϵdA by purified Polθ

To better understand the ability and mechanism of purified Polθ for TLS through ϵdA, we carried out in vitro DNA synthesis assays on DNA substrates that harbor a single ϵdA lesion with the (residues 1708–2590) WT Polθ protein and the Y2387A and Y2391A mutant Polθ proteins. For comparison, we also examined synthesis on DNA containing an AP site, in the form of a tetrahydrofuran (THF) moiety. The Polθ(1708–2590) protein affects TLS opposite the ϵdA and AP site similarly as the full-length Polθ (kindly provided by Richard Pomerantz).

We first performed assays with DNA substrates containing a running start primer, where DNA synthesis initiates 3 nt before the lesion (Fig. 2A). We analyzed DNA synthesis by (residues 1708–2590) WT Polθ and the Y2387A and Y2391A mutant Polθ proteins, each at three different protein concentrations (0.2, 1, and 10 nm) on undamaged DNA and on the ϵdA and AP site–containing DNA substrates. The Y2387A mutation exhibited a strong deleterious effect on DNA polymerase activity of Polθ. On undamaged DNA, DNA synthesis by 10 nm Polθ Y2387A protein was about the same as that for 0.2 nm WT Polθ, suggesting that it is at least ∼50-fold less efficient for polymerase activity. Importantly, Y2387A Polθ lacked the ability to incorporate a nucleotide opposite ϵdA or opposite an AP site even at high protein concentrations (Fig. 2A). On undamaged DNA, the Y2391A Polθ protein exhibited a moderate decline in DNA polymerase activity, but not as severe as the Y2387A Polθ. We estimate a reduction in catalytic efficiency of ∼10 fold, based on the similar DNA synthesis by 1 nm WT Polθ versus a 10 nm concentration of the Y2391A mutant. Even though Y2391A Polθ is less efficient in DNA synthesis, it inserts a nucleotide opposite ϵdA and the AP site. However, there is a complete lack of extension of synthesis opposite from either lesion (Fig. 2A).

Figure 2.

DNA polymerase activity of (residues 1708–2590) WT Polθ, Y2387A Polθ, or Y2391A Polθ on undamaged, ϵdA, or AP site-containing DNAs. A, increasing amounts of each protein were assayed with 10 nm template in the presence of 25 μm each of dGTP, dATP, dTTP, and dCTP for 5 min using the standard DNA polymerase assay conditions given under “Experimental procedures.” A diagrammatic representation of the DNA substrate is shown at the top, wherein the asterisk indicates the presence of an undamaged A, an ϵdA, or an AP lesion. Increasing protein amounts are indicted by triangles, and the concentrations for each set were 0.2, 1.0, and 10.0 nm. The positions of the 29-mer primer and the 52-nt full extension products are shown on the right. The position of the template A, ϵdA, or AP site, 4 nt 3′ to the primer terminus, is indicated by the asterisk on the right. B, nucleotide incorporation by WT Polθ, Y2387A Polθ, or Y2391A Polθ opposite an undamaged A, ϵdA, or an AP site. Assays were performed using the standard DNA polymerase conditions and contained a 25 μm concentration of either dCTP, dTTP, dGTP, or dATP, indicated by C, T, G, or A, or all four dNTPs combined, indicated by N. The protein concentration for each assay is given in parentheses, and all assays were carried out for 5 min, except those containing the Polθ Y2387A mutant protein, which were carried out for 10 min. A diagrammatic representation of the DNA substrate is shown at the top, wherein the asterisk indicates the presence of an undamaged A, an ϵdA, or an AP site. Positions of the primer and full-length product are shown on the right. The asterisk on the right indicates the position of the undamaged A, the ϵdA, or the AP site.

Next, we qualitatively assessed the fidelity of nucleotide incorporation opposite ϵdA by including only a single nucleotide in the assays, rather than all four. For these assays on undamaged DNA, we used 10-fold more mutant protein than WT protein because DNA synthesis is reduced by the Y2387A and Y2391A mutations. On undamaged DNA, WT Polθ incorporates T opposite A most efficiently, as do the Y2387A and Y2391A mutant proteins (Fig. 2B). In the presence of all four dNTPs, Y2387A also incorporates a C at about 20% compared with T. Y2391A Polθ is also error-prone, as indicated by the number of doublets and altered DNA ladder as compared with WT Polθ. Opposite ϵdA, the WT protein can incorporate A or G, but an A is incorporated the most, and in the presence of all four dNTPs, only an A is incorporated, and Polθ extends synthesis to the end of the template (Fig. 2B). At the same protein concentration, the Y2387A Polθ protein is unable to incorporate a nucleotide opposite the ϵdA or AP site. Opposite both the ϵdA and AP lesions, nucleotide incorporation by Y2391A Polθ is reduced compared with the WT protein; it primarily inserts a G, but an A is also inserted with a reduced proficiency. Also, as was seen in the running start assay (Fig. 2A), Y2391A Polθ is completely deficient in extending synthesis past ϵdA or the AP site (Fig. 2B).

Next, we examined the effects of Y2387A/Y2391A double mutation on DNA synthesis by Polθ on undamaged and ϵdA-containing DNAs (Fig. 3). In contrast to the individual Polθ Y2387A and Y2391A mutant proteins, the double Y2387A/Y2391A mutant Polθ is severely deficient in polymerase activity. When Polθ Y2387A/Y2391A is assayed on the undamaged DNA substrate at a 5-fold molar excess of protein over DNA, the polymerase only incorporates 4 nt (Fig. 3, lane 8), whereas the Polθ Y2387A single-mutant protein is able to synthesize up to ∼17 nt in assays containing equimolar protein:DNA concentrations (Fig. 2A, lane 7). Not surprisingly, on the ϵdA- and AP-containing DNA substrates, Polθ Y2387A/Y2391A behaves similarly to Polθ Y2387A, and no nucleotide incorporation is observed opposite either lesion. Thus, the reduced catalytic activity of the Polθ Y2387A/Y2391A mutant appears to be an additive effect of each of the Tyr to Ala mutations.

Figure 3.

DNA polymerase activity of (residues 1708–2590) WT Polθ or Y2387A/Y2391A Polθ on undamaged, ϵdA, or AP site-containing DNAs. Increasing amounts of each protein were assayed with 10 nm template in the presence of 25 μm each of dGTP, dATP, dTTP, and dCTP for 5 min using the standard DNA polymerase assay conditions given under “Experimental procedures.” A diagrammatic representation of the DNA substrate is shown at the top, wherein the asterisk indicates the presence of an undamaged A, an ϵdA, or an AP site. Increasing protein amounts are indicted by triangles. The concentrations for each set were 0.2, 1, and 10 nm for the WT protein and 0.2, 1, 10, and 50 nm for the Y2387A/Y2391A mutant derivative. The positions of the primer and the 52-nt full extension products are shown on the right. The position of the template A, ϵdA, or AP site, 4 nt 3′ to the primer terminus, is indicated by the asterisk on the right.

Tyr-2387 and Tyr-2391 are dispensable for Polθ-mediated TLS through ϵdA in human cells

Our findings, that the Y2387A and Y2391A mutations inactivate purified Polθ's ability to replicate through the ϵdA lesion and that there is a strong concordance in the pattern of TLS and nucleotide incorporation opposite the ϵdA and AP lesions by the purified WT Polθ and the Y2387A and Y2391A mutant Polθ proteins (Fig. 2), have suggested that the Tyr-2387 and Tyr-2391 residues modulate TLS through ϵdA, adopting an “AP” mode in the active site of purified Polθ. Thereby, by predominantly inserting an A opposite the adduct, purified Polθ conducts extremely error-prone TLS through ϵdA. In human cells, however, Polθ-mediated TLS through ϵdA is largely error-free, as the correct nucleotide T is inserted in over 90% of TLS products (12). Because T insertion opposite ϵdA could occur only if the adduct adopts a syn conformation and forms a Hoogsteen base pair with T(9), the Tyr-2387 and Tyr-2391 residues may play little or no role in TLS through ϵdA in human cells because these residues effect the “AP” mutagenic mode of TLS through ϵdA.

To determine the contribution of Tyr-2387 and Tyr-2391 residues to TLS in human cells, we analyzed the effects of Y2387A and Y2391A mutations in Polθ(1708–2590) on TLS opposite ϵdA carried on the leading-strand template of a duplex plasmid in which bidirectional replication ensues from a replication origin (Fig. 4). As shown in Table 1, in WT HFs expressing genomic Polθ, TLS opposite ϵdA occurs with a frequency of ∼25%. TLS is reduced to ∼14% in Polθ-depleted cells carrying the empty vector or carrying an siRNA-sensitive full-length WT Polθ. TLS is restored to WT levels in Polθ-depleted cells harboring siRNA-resistant WT Polθ(1708–2590). Thus, the effect of Polθ(1708–2590) on TLS opposite ϵdA is the same as that of genomically expressed Polθ. Importantly, in Polθ-depleted cells expressing Y2387A or Y2391A mutant Polθ (Fig. 5A), TLS occurs at WT levels (Table 1). In the absence of Polθ, TLS opposite ϵdA is performed primarily by the Polι/Polζ-dependent error-free pathway, which requires Rev1 as a scaffolding component, and by a relatively minor pathway, which requires Rev1 polymerase activity (12). Hence, in the absence of Rev1, both the Polι/Polζ and Rev1 polymerase-dependent pathways become inactive, and only the Polθ-dependent pathway remains functional, whereas in the absence Rev1 and Polθ, all of the TLS pathways are inactivated (12). In HFs co-depleted for Rev1 and Polθ where TLS would be abolished as indicated by the near absence of TLS in Rev1^−/− MEFs depleted for Polθ or in Polθ^−/− MEFs depleted for Rev1 (12) (see Table 2), expression of siRNA-resistant WT Polθ raises TLS to ∼11%, and importantly, expression of siRNA-resistant Y2387A or Y2391A mutant Polθ also raises TLS to WT Polθ levels (Table 1). Thus, in contrast to their indispensability for TLS by purified Polθ, the Y2387A or Y2391A mutations have no perceptible effect on TLS in human cells.

Figure 4.

TLS assay opposite ϵdA. A, chemical structure of ϵdA. B, in the SV40-based plasmid, a 16-mer target sequence (shown at the top) containing an ϵdA at A* is inserted between the BamHI and SbfI restriction sites in the lacz′ gene. The ϵdA-containing DNA strand is in-frame and therefore functional; it also carries the kan⁺ gene. TLS through the adduct generates Kan⁺ blue colonies.

Table 1.

Effects of Y2387A, Y2391A, or Y2387A/Y2391A Polθ mutations on TLS through the ϵdA adduct carried on the leading-strand DNA template in HFs (GM637)

siRNA	Vector expressing	Number of Kan+ colonies	Number of blue colonies among Kan+	TLS
				%
NC	Vector control	416	104	25.0
Polθ	Vector control	424	60	14.2
Polθ	FLAG-WT Polθ	388	54	13.9
Polθ	FLAG-siRa-WT Polθ	375	90	24.0
Polθ	FLAG-siR-Y2387A Polθ	405	103	25.4
Polθ	FLAG-siR-Y2391A Polθ	397	102	25.7
Polθ	FLAG-siR-Y2387A/Y2391A Polθ	364	100	27.5
Rev1+ Polθ	FLAG-siR-WT Polθ	378	40	10.6
Rev1+ Polθ	FLAG-siR-Y2387A Polθ	342	38	11.1
Rev1+ Polθ	FLAG-siR-Y2391A Polθ	366	36	9.8
Rev3+ Polθ	FLAG-siR-WT Polθ	286	35	12.2
Rev3+ Polθ	FLAG-siR-Y2387A Polθ	304	40	13.2
Rev3+ Polθ	FLAG-siR-Y2391A Polθ	312	42	13.5

^a siR, siRNA-resistant.

Figure 5.

Western blot analysis of Polθ expression. Shown are GM637 HFs (A) and Polθ^−/− MEFs (B) expressing 3xFLAG-WT Polθ(1708–2590), or the (residues 1708–2590), Y2387A, Y2391A, or Y2387A/Y2391A mutant Polθ or harboring the vector control. GM637 HFs were treated with Polθ siRNA for 48 h. Protein expression was determined by Western blotting with FLAG antibody (Sigma). β-Tubulin (Santa Cruz Biotechnology, Inc.) was used as the loading control.

Table 2.

Effects of Y2387A, Y2391A, or Y2387A/Y2391A Polθ mutations on TLS through the ϵdA adduct carried on the leading-strand DNA template in Polθ^−/− MEFs

Vector expressinga	Number of Kan+ colonies	Number of blue colonies among Kan+ colonies	TLS
			%
NC siRNA
No Polθ (control)	403	44	10.9
D570A/E571A mutant Polθ	308	31	10.1
WT Polθ	342	72	21.1
Y2387A Polθ	412	84	20.4
Y2391A Polθ	431	90	20.9
Y2387A/Y2391A Polθ	408	86	21.1
mRev1b siRNA
No Polθ (control)	422	5	1.2
D570A/E571A mutant Polθ	416	6	1.4
WT Polθ	367	32	8.7
Y2387A Polθ	411	38	9.2
Y2391A Polθ	428	40	9.3
Y2387A/Y2391A Polθ	402	40	10.0

^a Experimental procedures and Fig. 5B.

^b Mouse Rev1 siRNA.

In biochemical assays, Y2387A mutant Polθ is completely defective in TLS through ϵdA, whereas Y2391A mutant Polθ can insert a nucleotide opposite ϵdA but fails to extend synthesis (Fig. 2). That raised the possibility that in human cells, pursuant to nucleotide insertion opposite the lesion site by Y2391A Polθ, another polymerase extends synthesis. Because Polζ is a proficient extender of synthesis from the nucleotide inserted opposite the ϵdA lesion by Polι (9), and also opposite from a large variety of other distorting DNA lesions including the AP lesion (19, 20), we determined whether such a Polζ role could account for proficient Y2391A-mediated TLS in human cells. However, our findings that TLS occurs at the same level (∼13%) in HFs co-depleted for Rev3 and Polθ and expressing WT Polθ or the Y2387A or Y2391A mutant Polθ protein furnish clear evidence for the lack of any Polζ involvement (Table 1). Thus, although purified Y2391A mutant Polθ fails to extend synthesis from the nucleotide opposite ϵdA, this mutation imparts no impairment in TLS through ϵdA in human cells.

Next, we verified the effects of Y2387A and Y2391A mutations on TLS opposite ϵdA in Polθ^−/− MEFs. In Polθ^−/− MEFs harboring the vector or expressing catalytically inactive D570A/E571A mutant Polθ, TLS occurs at ∼10% (Table 2). Expression of WT Polθ raises the TLS level to ∼21%, and expression of the Y2387A or Y2391A mutant Polθ (Fig. 5B) also restores WT levels of TLS in Polθ^−/− MEFs (Table 2). In Polθ^−/− MEFs depleted for Rev1 and expressing either no Polθ or catalytically inactive D570A/E571A Polθ, TLS is almost completely abolished (∼1%), whereas expression of Y2387A or Y2391A mutant Polθ raises TLS to the same level (∼9%) as expression of WT Polθ (Table 2). Thus, both in HFs and MEFs, Y2387A and Y2391A mutations support TLS through ϵdA to the same extent as does WT Polθ.

Tyr-2387 is required for mutagenic TLS by Polθ opposite ϵdA in human cells

In human cells, Polθ replicates through ϵdA by incorporating the correct nucleotide T in over 90% of TLS products, and it also incorporates a C in ∼5% or an A in ∼3% of TLS products (12). Because Polθ and Rev1 polymerase activity contribute to alternative error-prone TLS pathways (12), in Polθ-depleted HFs carrying siRNA-sensitive full-length WT Polθ, mutagenic TLS emanating from Rev1 polymerase action occurs at a frequency of ∼11% (Table 3). Expression of siRNA-resistant WT Polθ raises mutagenic TLS to ∼15%, the increase in mutagenic TLS resulting from Polθ contribution (Table 3). Importantly, expression of Y2387A Polθ reduces mutagenic TLS to ∼6% (Table 3). Because error-prone TLS by Rev1 polymerase action would remain in these cells, this reduction in mutagenic TLS could have come about if Polθ's involvement in mutagenic TLS was inhibited by the Y2387A mutation. To confirm this possibility, we examined the frequency of mutagenic TLS in HFs co-depleted for Rev1 and Polθ and expressing Y2387A mutant Polθ (Table 3, last row). Our results that mutagenic TLS is abolished in these HFs concur with a role of Tyr-2387 in encumbering upon Polθ the capacity for mutagenic TLS opposite ϵdA.

Table 3.

Effects of Y2387A, Y2391A, or Y2387A/Y2391A mutations in Polθ on mutation frequencies and nucleotides inserted opposite the ϵdA adduct carried on the leading-strand DNA template in HFs (GM637)

siRNA	Vector expressing	No. of Kan+ blue colonies sequenced	Nucleotide inserted				Mutation frequency	Error-prone pathway that remains active
			A	G	C	T
							%
Polθ	FLAG-WT Polθ	146 (16)a	3	2	11	130	11.0	Rev1 Polb
Polθ	FLAG-siRc-WT Polθ	138 (21)	5	1	15	117	15.2	Rev1 Pol, Polθ
Polθ	FLAG-siR-Y2387A Polθ	144 (9)	1	0	8	135	6.3	Rev1 Polb
Polθ	FLAG-siR-Y2391A Polθ	156 (57)	20	10	27	99	36.5	Rev1 Pol, Y2391A Polθ
Polθ	FLAG-siR-Y2387A/Y2391A Polθ	136 (9)	1	0	8	127	6.6	Rev1 Polb
Rev1 + Polθ	FLAG-siR-Y2387A Polθ	108 (0)	0	0	0	108	0	None

^a The number of colonies in which TLS occurred by insertion of a nucleotide other than T is shown in parenthesis.

^b The greater reduction in mutation frequency in rows 3 and 5 than in row 1 is because of the increase in TLS frequency that occurs in cells expressing the Y2387A or the Y2387A/Y2391A Polθ (see Table I) and because of the ablation of Polθ-mediated mutagenic TLS by the Y2387A mutation.

^c siR, siRNA-resistant.

Next, we verified these observations in Polθ^−/− MEFs. As shown in Table 4, mutagenic TLS in Polθ^−/− MEFs, which would accrue from a Rev1 polymerase role, occurs at a frequency of ∼10%, and this frequency rises to ∼15% in cells expressing WT Polθ; by contrast, expression of Y2387A Polθ in Polθ^−/− MEFs reduces mutagenic TLS to ∼8%. Our results that in Rev1-depleted Polθ^−/− MEFs expressing WT Polθ, mutagenic TLS occurs at ∼7% (Table 4, fourth row from bottom) and that mutagenic TLS is abolished in Rev1-depleted Polθ^−/− MEFs expressing Y2387A Polθ (Table 4, third row from bottom) add further confirmatory evidence that Tyr-2387 confers upon Polθ the capability for mutagenic TLS in MEFs similar to that in HFs (Table 3).

Table 4.

Effects of Y2387A, Y2391A, or Y2387A/Y2391A mutations in Polθ on mutation frequency and nucleotides inserted opposite ϵdA carried on the leading-strand DNA template in Polθ^−/− MEFs

MEFs (siRNA)	Vector expressing	No. of Kan+ blue colonies sequenced	Nucleotide inserted				Mutation frequency	Error-prone pathway that remains active
			A	G	C	T
							%
Polθ−/−	Vector control	144 (15)a	3	1	11	129	10.4	Rev1Pol
Polθ−/−	WT Polθ	138 (20)	5	1	14	118	14.5	Rev1Pol, Polθ
Polθ−/−	Y2387A Polθ	140 (11)	2	0	9	129	7.9	Rev1 Pol
Polθ−/−	Y2391A Polθ	144 (40)	13	5	22	104	27.8	Rev1 Pol, Y2391A Polθ
Polθ−/−	Y2387A/Y2391A Polθ	152 (11)	2	0	9	141	7.2	Rev1 Pol
Polθ−/− (mRev1 siRNA)b	WT Polθ	96 (7)	4	0	3	89	7.3	Polθ
Polθ−/− (mRev1 siRNA)	Y2387A Polθ	96 (0)	0	0	0	96	0.0	None
Polθ−/− (mRev1 siRNA)	Y2391A Polθ	90 (18)	8	4	6	72	20.0	Y2391A Polθ
Polθ−/− (mRev1 siRNA)	Y2387A/Y2391A Polθ	104 (0)	0	0	0	104	0.0	None

^a The number of colonies in which TLS occurred by insertion of a nucleotide other than T is shown in parenthesis.

^b mRev1, mouse Rev1 siRNA.

Tyr-2391 affects suppression of mutagenic TLS by Polθ opposite ϵdA in human cells

In contrast to the effect of Y2387A mutation on the ablation of mutagenic TLS, the frequency of mutagenic TLS is elevated to ∼36% in Polθ-depleted HFs expressing Y2391A Polθ (Table 3). In Polθ^−/− MEFs expressing Y2391A Polθ, mutagenic TLS occurs at ∼28% (Table 4). Because mutagenic TLS conferred by both Rev1 polymerase and Y2391A Polθ would operate in these cells, we analyzed the frequency of mutagenic TLS in Polθ^−/− MEFs depleted for Rev1 and expressing Y2391A Polθ, because then only the contribution of Y2391A Polθ would remain. We find that mutagenic TLS occurs at ∼20% in these MEFs (Table 4, second row from bottom). This observation that Y2391A elevates Polθ-mediated mutagenic TLS implies a role of Tyr-2391 in the suppression of mutagenic TLS.

Epistatic interaction of Tyr-2391 with Tyr-2387 dampens Polθ mutagenicity opposite ϵdA in human cells

The abolition of mutagenic TLS by the Y2387A mutation and the enhancement of mutagenic TLS by the Y2391A mutation suggested that the Tyr-2387 and Tyr-2391 residues interact epistatically such that Tyr-2391 suppresses Tyr-2387 action in mutagenic TLS, and the observed frequency of ∼6–8% of mutagenic TLS by Polθ is sustained by that interaction. To explore this possibility, we analyzed the effects of the Y2387A/Y2391A double mutation on the frequency of TLS and its mutagenicity in Polθ-depleted HFs and in Polθ^−/− MEFs. Surprisingly, despite the severe defect in DNA synthesis by the purified enzyme (Fig. 3), the Y2387A/Y2391A mutant Polθ supports WT Polθ levels of TLS in HFs (Table 1) and in MEFs (Table 2). In Rev1-depleted Polθ^−/− MEFs expressing Y2387A/Y2391A Polθ, where only the Polθ function in TLS would remain, TLS occurs at WT Polθ rates (Table 2, last row), but mutagenic TLS is abolished (Table 4, last row). The abolition of mutagenic TLS by the Y2387A/Y2391A mutation is compatible with an epistatic interaction between Tyr-2387 and Tyr-2391, wherein Tyr-2387 effects mutagenic TLS and Tyr-2391 curtails Tyr-2387 action in mutagenic TLS.

Discussion

Evidence for adoption of a different configuration by the Polθ active site for TLS through ϵdA in human cells

The observation that similar to that opposite an AP site, purified Polθ predominantly inserts an A opposite ϵdA has suggested that Polθ replicates through ϵdA using an “AP” mode wherein ϵdA becomes extrahelical. In human cells, however, Polθ replicates through ϵdA by inserting the correct nucleotide T in over 90% of TLS products. Because ϵdA lacks the W-C edge (Fig. 4A), a T could be inserted opposite ϵdA only if the adduct adopts a syn conformation and forms a Hoogsteen base pair with the incoming T (Fig. 6). Hence, the Polθ active site must adopt a different configuration for mediating TLS in human cells than that in purified Polθ. Evidence from biochemical and genetic studies with mutations in the highly conserved Tyr-2387 and Tyr-2391 residues in the Polθ active site validates this hypothesis.

Figure 6.

Hoogsteen base pairing of ϵdA in syn with T, C, G, or an A in anti conformation. Dots, hydrogen bonds; R, sugar moiety.

In TLS assays with purified Polθ, Y2387A mutant Polθ lacks the capacity to insert a nucleotide opposite ϵdA, whereas Y2391A mutant Polθ primarily inserts a G and to a lesser extent an A (Fig. 2B), but it fails to extend synthesis further (Fig. 2). Similar to that seen with WT Polθ, the pattern of TLS and of nucleotide incorporation by mutant Polθ proteins opposite ϵdA resembles that opposite the AP lesion (Fig. 2). The complete inhibition of TLS by the Y2387A mutation opposite ϵdA and the AP lesion indicates that Tyr-2387 is indispensable for TLS opposite both the lesions, and the observation that Y2391A Polθ predominantly inserts a G and less well an A opposite both of the lesions suggests that in the absence of functional Tyr-2391, Tyr-2387 promotes the insertion of a G or an A but does not support extension.

In striking contrast to the indispensability of Tyr-2387 and Tyr-2391 for TLS by purified Polθ, mutational inactivation of these residues has no perceptible effect on TLS opposite ϵdA in HFs or MEFs; these mutations, however, affect the mutagenicity of TLS in HFs and MEFs. Mutational analyses of TLS products in WT HFs and in Rev1^−/− MEFs in a previous study (12) and in WT HFs and Polθ^−/− MEFs in this study show that whereas TLS mediated by WT Polθ generates ∼6–8% of mutational TLS products in which a C (∼5%) or an A or G (∼1–3%) are incorporated opposite ϵdA, the Y2387A mutation inhibits mutagenic TLS, and the Y2391A mutation increases the misincorporation of C, A, or G to ∼20% (Table 4). These results taken together with the observation that mutagenic TLS is also inhibited by the Y2387A/Y2391A double mutation (Table 4) suggest that the observed level of mutagenic TLS by WT Polθ (∼6–8%) in HFs and MEFs is attained by a mechanism in which Tyr-2387 promotes the misincorporation of nucleotide opposite ϵdA, whereas Tyr-2391 suppresses it.

The indispensability of Tyr-2387 and Tyr-2391 for TLS by purified Polθ (Fig. 2) but not for TLS in HFs and MEFs (Tables 1 and 2) implies that for mediating TLS through the ϵdA adduct, the roles of these highly conserved residues—important for DNA synthesis by the purified enzyme—are minimized in the Polθ active site reconfigured for TLS through ϵdA in human cells.

Mechanism of Polθ for replicating through ϵdA in human cells

The indispensability of Tyr-2387 for mutagenic TLS through ϵdA by purified Polθ and the requirement of this residue for Polθ-dependent mutagenic TLS in HFs and MEFs might suggest that mutagenic TLS in human and mouse cells operates by the same mechanism that the purified enzyme employs for replicating through ϵdA, wherein ϵdA adopts an “AP” mode. However, because a C is inserted opposite ϵdA in mutagenic TLS in WT HFs and MEFs, and because a C could be inserted only if ϵdA adopts a syn conformation and forms a Hoogsteen base pair with C in anti conformation (Fig. 6), Tyr-2387–mediated C insertion opposite ϵdA would occur via this mechanism. The adoption of syn conformation for C incorporation modulated by the Tyr-2387 residue would suggest that the incorporation of an A or a G opposite ϵdA by Tyr-2387 also occurs by Hoogsteen pairing between ϵdA in syn conformation and an A or G in anti conformation (Fig. 6). Thus, the mechanism of Hoogsteen base pairing via which Tyr-2387 and Tyr-2391 coordinate the incorporation of C, A, or G opposite ϵdA in HFs and MEFs would differ from the mechanism of adopting an AP-like mode that purified Polθ employs for misincorporating A opposite ϵdA. And importantly, the predominant incorporation of T opposite ϵdA (∼92%) could only occur by the adoption of syn conformation by ϵdA in the Polθ active site (Fig. 6).

Possible mechanism for reconfiguration of the Polθ active site for TLS through ϵdA in human cells

The lack of requirement of the Tyr-2387 and Tyr-2391 residues for the predominant error-free TLS through ϵdA in human and mouse cells and the proposal that even the mutagenic TLS that depends upon these residues would entail the adoption of a syn conformation by ϵdA in the Polθ active site can be rationalized if the Polθ active site adopts a different configuration for TLS in human cells than in purified Polθ. To explain the acquisition of a different configuration in the Polθ active site, we posit that Polθ functions in TLS in human cells as a component of a multiprotein ensemble and that protein-protein interactions and post-translational modifications in the components of this ensemble modulate the Polθ active site such that it promotes rotation of ϵdA into a syn conformation, allowing for Hoogsteen base pairing with the incoming nucleotide.

In the Polθ active site reconfigured for conducting predominantly error-free TLS through ϵdA in human cells, the roles of Tyr-2387 and Tyr-2391 residues become much less eminent, affecting only the mutagenic TLS. In the structure of purified Polθ, Tyr-2387 participates in hydrogen-bonding to the β-phosphate of the incoming dNTP, and Tyr-2391 forms part of the active-site floor beneath the template residue (18). This explains the requirement of these residues for efficient DNA synthesis on undamaged DNA and for TLS through ϵdA by the purified enzyme (Figs. 2 and 3). By contrast, the lack of their requirement for predominantly error-free TLS through ϵdA in human cells implies that in the reconfigured Polθ active site, these residues no longer affect the stabilization of the template or the incoming nucleotide for incorporation of the correct dNTP.

TLS Pols, such as η, ι, κ, or Rev1 have a preformed active site adapted for replicating through specific types of DNA lesions. In these Pols, the action mechanisms stay the same for TLS in human cells as those indicated from biochemical and structural studies of the purified Pol, as, for example, in the role of Polη in TLS opposite CPDs and in the role of Polι in TLS opposite ϵdA. Replicative DNA Pols also utilize similar action mechanisms in vitro and in vivo. Thus, among DNA Pols, Polθ provides the first example where the action mechanism for TLS in human cells differs from the mechanism adopted by the purified enzyme.

Experimental procedures

Polθ expression in yeast

The human Polθ(1708–2590) protein harboring the catalytically active C-terminal DNA polymerase domain was expressed as a fusion with glutathione S-transferase (GST) from plasmid pPOL507 as described (21). The Y2387A and Y2391A mutations were each generated by PCR using mutagenic oligomers and the Polθ(1708–2590) cDNA in pPOL523 as template. The mutant cDNAs were fully sequenced to confirm the presence of the mutations and were cloned into the expression vector, generating plasmids pJR65, pPOL665, and pBJ2333, which express GST-tagged Polθ(1708–2590) Y2387A, Polθ(1708–2590) Y2391A, and Polθ(1708–2590) Y2387A/Y2391A, respectively.

WT and mutant Polθ(1708–2590) expression plasmids were transformed into yeast strain YRP654, and the proteins were expressed and affinity-purified using GSH Sepharose as described (22). The GST fusion tag was removed from each Polθ(1708–2590) protein by treatment with prescission protease, leaving a 7-amino acid linker attached to the N terminus of Polθ. Proteins were quantified by densitometry of Coomassie-stained protein samples separated by 11% SDS-PAGE using ImageQuant software (GE Biotech).

DNA polymerase assays

The standard DNA polymerase assay (5 μl) contained 25 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 1 mm DTT, 10% glycerol, 0.1 mg/ml BSA, and DNA substrate. The DNA substrates consisted of a ³²P-5′-labeled DNA primer annealed to a 52-mer template with the sequence 5′-TTCGTATA ATGCCTAC ACT[A]GAGT ACCGGAGC ATCGTCGT GACTGGGA AAAC-3′, in which [A] at position 20 indicates either an undamaged A, ϵdA, or a THF moiety (AP site analog). The ϵdA- and THF-containing templates were synthesized by the Midland Certified Reagent Company (Midland, TX) and were PAGE-purified. For running start assays, the 29-mer oligonucleotide primer 5′-GTTTTCCCAG TCACGACGAT GCTCCGGTA-3′ was annealed to each template. To assay nucleotide incorporation opposite A, ϵdA, or the AP site, the 23-mer primer 5′-GTCACGACGATGCTCCGGTACTC-3′ was used. Single dNTPs, dATP, dGTP, dTTP, dCTP, or all four dNTPs combined were included at concentrations indicated in the figure legends. Reactions were initiated by the addition of 1 μl of DNA polymerase in 5× reaction buffer (125 mm Tris-HCl, pH 7.5, 5 mm DTT, 0.5 mg/ml BSA) and carried out at 37 °C for times indicated in the figure legends before termination by 6 volumes of 95% formamide loading buffer containing 0.06% xylene cyanol, 0.06% bromphenol blue. Reaction products were separated by 12 or 20% TBE, 8 m urea-PAGE. Gels were fixed in 10% methanol, 10% acetic acid for 10 min and dried, and products were visualized by phosphorimaging on a Typhoon FLA7000 (GE Biotech).

Construction of ϵdA plasmid vectors and TLS assays

The in-frame target sequence of the lacZ′ gene containing the ϵdA lesion is shown in Fig. 4. The detailed methods for construction of lesion-containing SV40 duplex plasmid, for TLS assays, and for mutational analysis of TLS products have been described previously (15, 23).

Stable expression of WT Polθ and mutant Polθ in HFs or MEFs

DNAs encoding human WT Polθ(1708–2590) or the mutant (residues 1708–2590) Y2387A, Y2391A, or Y2387A/Y2391A Polθ, respectively, were cloned into vector pCMV7–3xFLAG-zeo (Sigma). The resulting vectors were transfected into normal human fibroblast (GM637) cells or Polθ^−/− MEF cells by iMFectin transfection reagent (GenDEPOT). After a 24-h incubation, 0.5 μg of Zeocin (GenDEPOT) were added to the culture medium. After 3 days of incubation, cells were washed with PBS buffer and were continuously cultured with the medium containing 250 ng of Zeocin for ∼2 weeks. Protein expression and siRNA knockdowns were checked by Western blot analysis (Fig. 5) as described before (13).

Data availability

All of the data are contained within the article.

Author contributions

J.-H. Y. and S. P. formal analysis; J.-H. Y. and R. E. J. validation; J.-H. Y. and R. E. J. investigation; J.-H. Y. and R. E. J. methodology; R. E. J., L. P., and S. P. writing-review and editing; L. P. resources; L. P. and S. P. supervision; L. P. funding acquisition; L. P. and S. P. project administration; S. P. conceptualization; S. P. writing-original draft.