Biology subjects

Sugawara, N.

Publications and source records attributed to Sugawara, N..

4 recordsLinked to original sources

bioRxiv2025

Mutations and structural variants arising during double-strand break repair

Double-strand break (DSB) repair is highly mutagenic compared to normal replication. In budding yeast, repair of an HO endonuclease-induced DSB at MAT can be repaired by using a transcriptionally silent HMR::Kl-URA3 donor. During repair, -1 deletions in homonucleotide runs are strongly favored over +1 insertions, whereas during replication, spontaneous +1 and -1 events are equal. Microhomology-bounded, repair-associated intragenic deletions (IDs) are recovered 12 times more frequently than tandem duplications (TDs). IDs have a mean length of 56 bp, while TDs average 22 bp. These data suggest a picture of the structure of the repair replication fork: IDs and TDs occur within the open structure of a migrating D-loop, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either [~]80 bp ahead or [~]40 bp behind the 3 end. Another major class of repair-associated mutations ([~]10%) are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence in HMR is only 72% identical (homeologous) with S. cerevisiae ura3-52. ICTS events begin and end at regions of short ([~]7 bp) microhomology; however, ICTS events are constrained to the middle of the copied sequence. Whereas microhomology usage in intragenic deletions is not influenced by adjacent homeology, we show that extensive pairing of adjacent homeology plays a critical role in ICTS. Thus, although by convention, structural variants are characterized by the precise base pairs at their junction, microhomology-mediated template switching actually requires alignment of extensive adjacent homeology. Significance statementDNA synthesis during repair of a double-strand chromosome break by homologous recombination exhibits a high rate of mutation compared to normal replication. Using a genetic system in budding yeast, we isolated thousands of mutations occurring during repair. We conclude that the repair replication fork appears to have the two DNA strands open [~]80 bp ahead of the DNA polymerase, but the strands re-anneal rapidly behind the polymerase. Additionally, we analyzed interchromosomal template switching, in which the partially copied DNA strand dissociates and pairs with a new template at a short stretch of perfectly matching bases (microhomology), and resumes copying. We show that these apparent microhomology-mediated template switching events in fact require the pairing of [~]200 bp of imperfectly matching bases (homeology).

genetics↗

bioRxiv2023

Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork

Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MAT locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3- mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are -1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and -1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase {delta} copies the template, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3 end, within the open structure of a migrating D-loop. Our data suggest that [~]100 bp ahead of the polymerase is "open," but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.

genetics↗

bioRxiv2023

Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially-copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MAT, repaired by an HMR::Kl-URA3 donor. We inserted into HMR::Kl-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield mat::Kl-ura3::QP+4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP+4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP+4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (pol3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a +1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura3+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

genetics↗

bioRxiv2022

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.3-kb of phage lambda DNA in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3 and Slx4. A natural 3% divergence between repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL) are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the identical repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain, where the F variant carries six single-base mutations and one +1(T) insertion within a run of 10 Ts showed a gradient of correction favoring the sequence opposite each 3 end of the annealed strand. Mismatches (MMs) located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, using either 3 nonhomologous tail removal or 3 to 5 "proofreading" resection by DNA polymerase {delta} followed by synthesis to fill in the gap. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat, with a bias in favor of the "left" copy. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, while the deletion of MSH6 resulted in unrepaired MMs.

genetics↗