Minding the gap: the underground functions of BRCA1 and BRCA2 at stalled replication forks

Abstract

The hereditary breast and ovarian cancer predisposition genes, BRCA1 and BRCA2, participate in the repair of DNA double strand breaks by homologous recombination. Circumstantial evidence implicates these genes in recombinational responses to DNA polymerase stalling during the S phase of the cell cycle. These responses play a key role in preventing genomic instability and cancer. Here, we review the current literature implicating the BRCA pathway in HR at stalled replication forks and explore the hypothesis that BRCA1 and BRCA2 participate in the recombinational resolution of single stranded DNA lesions termed "daughter strand gaps", generated during replication across a damaged DNA template.

Fig. 1

Models of fork restart in eukaryotic cells. (A) Generation of DSBs at stalled forks. Leading strand lesion stalls the fork, which can undergo fork reversal to form a “chicken foot” (Holliday Junction) structure. The HJ is then processed to form a DSB. RecBCD-like functions then load Rad51, catalyzing invasion of the neighboring intact sister chromatid to form a D-loop structure. A eukaryotic PriA-like activity might prime DNA synthesis on the free 3′ end of the D-loop to restart replication in an origin independent manner. (B) Formation of a lagging strand DSG: lagging strand DNA polymerase stalling need not interrupt fork progression. (C) Formation of a leading strand DSG: the lagging strand polymerase may become uncoupled from the stalled leading strand polymerase, leaving a ssDNA gap on the leading strand. A PriC-like activity might reinitiate leading strand synthesis, leaving a DSG on the leading strand. Parental strands are labeled blue. Daughter strands are labeled red. Free 3′ DNA ends are marked with an arrowhead. DNA polymerase stalling lesion is indicated by a black square.

Fig. 2

HR mediated daughter strand gap repair. (A) Figure shows a leading strand DSG. RecFOR-like loading of Rad51 facilitates strand exchange with the sister chromatid without a DSB intermediate. Branch migration switches the polymerase stalling lesion to the donor sister, allowing continued synthesis of the previously blocked nascent strand. Double Holliday Junction dissolution by the Bloom’s syndrome helicase may preferentially generate non-crossover products [159]. (B) Model depicting possible competition between TLS polymerase (green hexagon) and RecFOR-like HR functions (orange circle) for DSG repair. Figure shows a lagging strand DSG. TLS and Rad51 loading are initiated at opposite ends of the DSG. Purple circles, RPA trimer; blue circles, Rad51 oligomerizing on ssDNA. Black arrows depict direction of polymerase elongation by TLS or of Rad51 nucleation on ssDNA. DNA polymerase stalling lesion is indicated by a black square. FA proteins may also regulate this choice [160].

Fig. 3

Mechanism of gene conversion by synthesis dependent strand annealing (SDSA) and break-induced replication (BIR). (A) In the SDSA model, following end processing, the 3′ end invades the sister chromatid and initiates repair synthesis without necessarily involving lagging strand synthesis. Repair synthesis copies a small stretch of information, after which the nascent strand is displaced and pairs with ssDNA of complementary sequence on the second end of the DSB. B. In the BIR model, a replication fork is established at the recombination joint, initiating both leading and lagging strand synthesis. Free 3′ DNA ends are marked with an arrowhead. Newly synthesized DNA is depicted by a dotted red line.

Fig. 4

Proposed roles of BRCA1 and BRCA2 in daughter strand gap repair. BRCA1/BARD1 (light blue) performs transcription/silencing functions on chromatin in the undamaged cell. An RPA-coated DSG (shown here on the lagging strand; RPA not shown) recruits Atr/AtrIP (red) and the 9-1-1 complex (green). Recruitment of BRCA1/BARD1 to DSGs may be independent of Atr. Activated Atr phosphorylates BRCA1, promoting interaction of BRCA1/BARD1 with other DNA damage response proteins (not shown), as discussed in the text. The BRCA1/BARD1 heterodimer may ubiquitylate target proteins at the DSG, and facilitates recruitment of the BRCA2/Rad51 complex (orange) for HR-mediated DSG repair. Rad51 is shown in dark blue. DNA polymerase stalling lesion is indicated by a black square. The HR function of BRCA1 depicted here is independent of the H2AX response [48].