BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids

Abstract

BRCA1 controls early steps of the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination, but has no known role following Rad51-mediated synapsis. Here we show that BRCA1 influences post-synaptic homologous recombination events, controlling the balance between short- (STGC) and long-tract gene conversion (LTGC) between sister chromatids. Brca1 mutant cells reveal a bias towards LTGC that is corrected by expression of wild type but not cancer-predisposing BRCA1 alleles. The LTGC bias is enhanced by depletion of CtIP but reversed by inhibition of 53BP1, implicating DNA end resection as a contributor to the STGC/LTGC balance. The impact of BRCA1/CtIP loss on the STGC/LTGC balance is abolished when the second (non-invading) end of the break is unable to support termination of STGC by homologous pairing (“annealing”). This suggests that BRCA1/CtIP-mediated processing of the second end of the break controls the annealing step that normally terminates SDSA, thereby suppressing the error-prone LTGC outcome.

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…However, crossing over is suppressed in somatic HR and, where analyzed, both I-SceI-induced and Tus/Ter-induced "long tract"-type HR products have been found to be the product of LTGC (i.e., extensive copying from the donor sister chromatid), rather than crossing over. 15,17,19,20 We found previously that increasing the number of Ter sites between 1 and 6 within the HR reporter produces progressively higher levels of Tus/Ter-induced HR, the largest array studied previously containing an array of 6 Ter elements. 15 To determine the impact on Tus/Ter-induced HR of increasing the Ter array size beyond 6, we generated reporters containing arrays of 9, 15 or 21 Ter repeats, each with an adjacent I-SceI site ( Fig.…”

“…We targeted each reporter, in parallel, to the ROSA26 locus of mouse ES cells carrying one conditional allele of BRCA1 and one mutant BRCA1 allele that inactivates the tandem BRCT repeat of the gene product to generate Brca1 fl/BRCT Ter/HR cells. 33 (These cells behave as if wild type with regard to BRCA1 15,20 ) We used Southern blotting and PCR to identify clones of each type containing only one intact, ROSA26-targeted HR reporter ( Fig. S1 and Materials and Methods).…”

“…15,20 Briefly, 2 mutant copies of the cDNA encoding enhanced green fluorescent protein ("GFP") are placed in tandem within the HR reporter. One GFP copy is full length but is interrupted by a Ter array followed by an I-SceI target site (Fig.…”

“…15 LTGC is an error-prone, non-crossover outcome of HR, entailing copying of several kilobases from the donor sister chromatid prior to HR termination, thereby generating a tandem duplication at the site of repair. 17,19,20 LTGC shares several properties with break-induced replication (BIR), a known mediator of genomic instability in yeast. [27][28][29][30][31] Indeed in BRCA1 mutant cells, LTGC is the most abundant HR product observed at Tus/Ter-stalled forks.…”

“…15 An alternative, aberrant HR outcome termed "long tract" gene conversion (LTGC) entails the copying of several kilobases from the donor sister chromatid and is considered an error-prone outcome. [17][18][19][20] One cause of LTGC can be a failure of termination of conventional HR, when the second (non-invading) DNA end is either missing-as might occur in a "one-ended" break-or imperfectly coordinated with the invading strand. 20 Analysis of Tus-Ter interactions in vitro showed that partial strand separation at the non-permissive end of Ter-such as might occur when the replicative helicase stalls within Terallows a cytosine at position 6 (C6) to flip into a pocket of Tus, thereby "locking" Tus onto Ter.…”