e RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements. R eplication fork maintenance is essential for maintaining the structural integrity of chromosomes, and replication defects were proposed to cause copy number variation (CNV) and complex genomic rearrangements (CGRs) (8,35). CNV is a natural change in the number of copies of one or more sections of DNA in the genome of a population ranging from one to thousands of kilobases and occurs between repeat segments (22, 23). CNV accounts for about 12% of the human genome and is important for murine (11) and primate (5, 46) evolution. CGRs cause genomic disorders and cancer in humans (13,23,74,75). They consist of at least two rearrangements with multiple breakpoints all closely aligned in the same chromosomal region, suggesting they derived from a single event. The genesis of structural chromosomal rearrangements is not known, yet compromised replication fork progression may evoke novel error-prone mechanisms, such as fork stalling and template switching (FoSTeS) (32) or microhomology-mediated break-induced replication (MMBIR) (22), which suppress broken replication forks but at the risk of structurally rearranging the genome. FoSTeS and MMBIR are not well understood at a mechanistic level but were proposed after sequencing of CGRs found in human genomic disorders and cancers. The sequence information showed that these events involved multiple chromosome segments and small levels of homology at some of the rearrangement junctions (22, 23). Therefore, defects in replication fork maintenance may promote CGRs and CNV.RAD51 is a RecA recombinase that is essential for replication fork maintenance. RAD51 performs two functions to suppress broken replication forks. First, RAD51 enables replication restart when a replica...

Replication fork (RF) maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation1-3. Potential causal mechanisms are homologous recombination (HR) and error-free postreplication repair (EF-PRR). HR repairs damage induced DNA double strand breaks (DSBs) and single-ended DSBs within replication. To facilitate HR, the recombinase RAD51 and mediator BRCA2 form a filament on the 3’ DNA strand at a break to enable annealing to the complementary sister chromatid4 while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination5. HR also stabilizes6,7 and restarts8,9 RFs without a DSB10,11. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6/RAD18 and UBC13/MMS2/RAD5 ubiquitin ligase complexes12. Some components are common to both HR and EF-PRR like RAD51 and RAD1813,14. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched repeats. By contrast, UV light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3’→5’ exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to UV light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed RF stalling in response to nucleotide depletion. Interestingly, RF stalling induced fusion for identical and mismatched repeats implicating faulty replication as a causal mechanism for both pathways.

BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to respectively facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3′ overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2lex1/lex2) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.

Fanconi anemia (FA) patients exhibit bone marrow failure, developmental defects and cancer. The FA pathway maintains chromosomal stability in concert with replication fork maintenance and DNA double strand break (DSB) repair pathways including RAD51-mediated homologous recombination (HR). RAD51 is a recombinase that maintains replication forks and repairs DSBs, but also rearranges chromosomes. Two RecQ helicases, RECQL5 and Bloom syndrome mutated (BLM) suppress HR through nonredundant mechanisms. Here we test the impact deletion of RECQL5 and BLM has on mouse embryonic stem (ES) cells deleted for FANCB, a member of the FA core complex. We show that RECQL5, but not BLM, conferred resistance to mitomycin C (MMC, an interstrand crosslinker) and camptothecin (CPT, a type 1 topoisomerase inhibitor) in FANCB-defective cells. RECQL5 suppressed, while BLM caused, breaks and radials in FANCB-deleted cells exposed to CPT or MMC, respectively. RECQL5 protected the nascent replication strand from MRE11-mediated degradation and restarted stressed replication forks in a manner additive to FANCB. By contrast BLM restarted, but did not protect, replication forks in a manner epistatic to FANCB. RECQL5 also lowered RAD51 levels in FANCB-deleted cells at stressed replication sites implicating a rearrangement avoidance mechanism. Thus, RECQL5 and BLM impact FANCB-defective cells differently in response to replication stress with relevance to chemotherapeutic regimes.

TREX2 is a 3′→5′ exonuclease that binds to DNA and removes 3′ mismatched nucleotides. By an in vitro structure function analysis, we found a single amino acid change (H188A) completely ablates exonuclease activity and impairs DNA binding by about 60% while another change (R167A) impairs DNA binding by about 85% without impacting exonuclease activity. For a biological analysis, we generated trex2 null cells by deleting the entire Trex2 coding sequences in mouse embryonic stem (ES) cells. We found Trex2 deletion caused high levels of Robertsonian translocations (RbTs) showing Trex2 is important for chromosomal maintenance. Here we evaluate the exonuclease and DNA binding domains by expressing in trex2 null cells coding sequences for wild type human TREX2 (Trex2 hTX2 ) or human TREX2 with the H188A change (Trex2 H188A ) or the R167A change (Trex2 R167A ). These cDNAs are positioned adjacent to the mouse Trex2 promoter by Cre-mediated knock-in. By observing metaphase spreads, we found Trex2 H188A cells exhibited high levels of double-strand breaks (DSBs) and chromosomal fragments. Therefore, TREX2 may suppress spontaneous DSBs or exonuclease defective TREX2 may induce them in a dominate-negative manner. We also found Trex2 hTX2 , hTrex2 H188A and hTrex2 R167A cells did not exhibit RbTs. Thus, neither the exonuclease nor DNA binding domains suppress RbTs suggesting TREX2 possesses additional biochemical activities.