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. 2020 Jan 10;11(1):79. doi: 10.3390/genes11010079

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Assays to study gross chromosomal rearrangements at inverted repeats. (A) An assay to study fork stalling at repetitive elements. (1) The RTS1 pause site was placed on the right of a functional ura4+ gene (uraR), (2) left (Rura) or (3) both sides (RuraR). Fork stalling was assayed using pulse field gel electrophoresis (PFGE). (B) Chromosomal rearrangements of constructs in (A) can occur by two mechanisms: gene conversion without crossover and gene conversion with crossover. In both cases there is a recombination event between the RTS1 site next to the ura4⁺ cassette and the endogenous RTS1 at the MAT locus on Ch.II. Both types of events result in loss of ura4⁺. (C) Modification of construct in (A) to monitor chromosomal dynamics by fluorescence microscopy. A LacO array was placed on the telomere side of the RTS1/ura4⁺ cassettes. (D) Repetitive elements constructs to study chromosomal rearrangements. (1) RTS1 cassettes were placed on both sides of two directional ura4⁺ repeats (Ru(dir)uR). (2) RTS1 was placed on both sides of inverted ura4⁺ repeats spaced by 14 bp of unique sequence (RuiuR). A construct without the 14bp spacer was also made (RuuR) (3) In oRuiuR the RTS1 is in opposite direction from the RuiuR construct. (4) In the RuhR construct, one of the ura4⁺ repeats was replaced with his3⁺. (E) Several constructs to test the effect of repeat length and the position of the replication pause sites in producing rearrangements. (1) Two ura4⁺ inverted repeats spaced by 14 bp of unique sequence were engineered with 0.2 Kb (Rpal1R) or 1.8 Kb (Rpal2R) his3⁺ sequence between the right ura4⁺ repeat and the RTS1 pause site. An additional short ura4⁺ was placed between the left RTS1 and the ura4⁺ repeat (yellow arrow). (2) Tpal1R and Tpal2R constructs are similar to constructs in (1) except that the left RTS1 was replaced with three TER2/3 sequences. (3) In this construct the right ura4⁺ repeat was replaced by a unique sequence. (4) Each of the ura4⁺ repeat sizes were varied between 30–1200 base pairs. (5) The spacer between the inverted repeats was varied between 7–250 base pairs. (6) ura4⁺ cassette flanked by the TER2/3 termination sites. (7). Direct repeats but one of the ura4⁺ repeats was replaced with a ura5⁺ repeat. (F) The ura4⁺ and RTS1 pause sites were placed next to known origins of replication (3004, 3005, 3006/7). (1) Co-directional ura4⁺ and RTS1 with RTS1 placed either after (1) or in front (2) of the ura4⁺ repeat. (3) Construct similar to (A) (2) and (4) construct similar to (A) (3) placed between the 3005 and 3006/7 replication origins. (5) Construct similar to (4) but with RTS1 in the opposite orientation. (G) Constructs to test microhomology mediate repair caused by rescued replication forks. (1) Two different microhomology constructs. ura4-dup20/ura4sd20 was engineered by placing two 20 bp (grey boxes) repeats flanked by 5 bp of microhomology sequences (black boxes) in the middle of the ura4⁺ gene. ura4-dup22/ura4sd22 is has 22 bp repeats flanked by 4 bp of homology. Microhomology mediated repair was investigated either in the absence of a pause site (2) or with the pause site placed on the left (3) or right (4) of the construct. Only ura4sd20 is shown.