Characteristics and possible mechanisms of formation of microinversions distinguishing human and chimpanzee genomes

Genomic inversions come in various sizes. While long inversions are relatively easy to identify by aligning high-quality genome sequences, unambiguous identification of microinversions is more problematic. Here, using a set of extra stringent criteria to distinguish microinversions from other mutational events, we describe microinversions that occurred after the divergence of humans and chimpanzees. In total, we found 59 definite microinversions that range from 17 to 33 nucleotides in length. In majority of them, human genome sequences matched exactly the reverse-complemented chimpanzee genome sequences, implying that the inverted DNA segment was copied precisely. All these microinversions were flanked by perfect or nearly perfect inverted repeats pointing to their key role in their formation. Template switching at inverted repeats during DNA replication was previously discussed as a possible mechanism for the microinversion formation. However, many of definite microinversions found by us cannot be easily explained via template switching owing to the combination of the short length and imperfect nature of their flanking inverted repeats. We propose a novel, alternative mechanism that involves repair of a double-stranded break within the inverting segment via microhomology-mediated break-induced replication, which can consistently explain all definite microinversion events.

The structure-specific endonuclease complex SLX4/XPF regulates Tus/Ter-induced homologous recombination

Vertebrate replication forks arrested at an interstrand DNA crosslink (ICL) can engage the Fanconi anemia (FA) pathway of ICL repair. The FANCP product, SLX4, binds the FANCQ/XPF/ERCC4-ERCC1 endonuclease, which incises bidirectionally arrested forks to ‘unhook’ the ICL. The resulting double strand break (DSB) is repaired by homologous recombination (HR). Whether this mechanism operates at replication blocks other than ICLs is unknown. Here, we study the role of mammalian SLX4 in HR triggered by a site-specific, chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus/Ter complex. We identify an SLX4-XPF-mediated step that is required for Tus/Ter-induced HR but not for HR induced by a replication-independent DSB. We additionally identify a requirement for SLX4-XPF in DSB-induced ‘long tract’ gene conversion, a replicative HR pathway related to break-induced replication. Our work suggests that Tus/Ter-induced HR recapitulates the incision step of replication-coupled ICL repair, and that the full FA mechanism can process DNA-protein barriers for HR.

Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome

The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.

Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.

Non-canonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast

Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field (CHEF) electrophoresis, next-generation whole genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or “late” template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.

The stabilized Pol31–Pol3 interface counteracts Pol32 ablation with differential effects on repair

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3–Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31–Pol3 and Pol31–Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells

Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.