Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats

Instability of the human minisatellite CEB1 in rad27Δ and dna2-1 replication-deficient yeast cells

Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them. Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations. We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Δ cells. Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Δ cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit. Altogether, these results suggest that the improperly processed 5′ flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events. This replication-dependent phenomenom differs from the recombination-induced instability in yeast meiotic cells

Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks.

International audienceMinisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models

Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates

This article was made OA through RCUK block grant funds.Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.Publisher PDFPeer reviewe

Mécanismes et contrôles de la recombinaison méiotique chez la levure Saccharomyces cerevisiae

Les mécanismes moléculaires de la recombinaison méiotique commencent à être mieux compris grâce à une série de travaux récents chez Saccharomyces cerevisiae. Il a été montré que la recombinaison méiotique est initiée par la formation de cassures double-brin de l’ADN qui peuvent être mises en évidence physiquement dans les premières heures de la méiose. Ces cassures double-brin sont localisées préférentiellement dans les régions intergéniques contenant un promoteur de transcription et sont regroupées dans des domaines le long des chromosomes. La protéine Spo11 qui est apparentée à une nouvelle famille de topoisomérases de type II, est très probablement la protéine responsable de la formation de ces cassures doublebrin. Plusieurs intermédiaires du processus de réparation des cassures double-brin ont été détectées physiquement chez la levure et des complexes multiprotéiques qui interviennent à différentes étapes au cours de la réparation ont été identifiés. Ces complexes multiprotéiques sont retrouvés chez les eucaryotes supérieurs, ce qui suggère une conservation des mécanismes de recombinaison méiotique. L’utilisation combinée de la génétique, de la cytologie, de la biochimie et des nouvelles technologies développées pour les études génomiques (puces à ADN) devrait aboutir à une meilleure compréhension des mécanismes de la recombinaison méiotique