Homologous Recombination and Replication Fork Protection: BRCA2 and More!

Abstract

BRCA2 is a breast and ovarian tumor suppressor that guards against genome instability, a hallmark of cancer. Significant progress has been made in improving our understanding of BRCA2 function from biochemical, cellular, and mouse studies. The knowledge gained has been actively exploited to develop therapeutic strategies, including PARP inhibition, which has shown promising clinical outcomes. Recently, tremendous excitement has been generated by the findings of the roles of BRCA2 and other proteins in suppressing replication stress through homologous recombination and in the protection of stalled replication forks. Processes such as mitotic DNA synthesis and fork reversal have taken center stage in these studies. Here, we discuss our recent findings in the context of these advances.

Figure 1.. HR and FP functions of BRCA2 to protect genome integrity.

(Left) HR repair of a DSB is initiated by resection of the break ends to generate 3’ single-stranded DNA overhangs. Subsequent strand invasion into a homologous DNA is critical for repair DNA synthesis, which ultimately promotes an error-free repair outcome. While RAD51 is crucial for strand invasion, BRCA2 plays an essential role by recruiting RAD51 onto the resected DNA. (Right) In the FP process, BRCA2 prevents the nascent strands of a stalled replication fork from being degraded by nucleases such as MRE11.

Figure 2.. Consequences of BRCA2 deficiency in multiple cell cycle phases.

As proposed in our recent study using MCF10A cells (Feng and Jasin 2017), even in unperturbed situations, BRCA2 deficiency causes replication stress that compromises the timely completion of DNA replication. The resulting under-replicated DNA leads to G2 DNA lesions and single-stranded DNA (ssDNA) formation, which in turn leads to abnormalities in mitosis and 53BP1 nuclear body formation in the subsequent G1 phase. Such G1 lesions trigger p53-dependent G1 arrest and cellular senescence as well as p53-independent apoptosis, resulting in cell inviability. At the functional level, suppression of replication stress is primarily mediated by the HR, rather than the FP, activity of BRCA2. (Reprinted from (Feng and Jasin 2017) under a Creative Commons license http://creativecommons.org/licenses/by/4.0/.)

Figure 3.. Mechanisms of fork degradation and protection.

(A) Fork degradation occurs on a reversed fork. Reversal of stalled replication forks is promoted by RAD51 recombinase and DNA translocases (SMARCAL1, ZRANB3, HLTF). In the absence of FP factors like BRCA2, MRE11 and other nucleases can lead to fork degradation. Different pathways regulate MRE11 and MUS81 recruitment to stalled forks. MRE11 recruitment is promoted by a number of proteins, including PARP1 and the PTIP-MLL3/4 axis, while MUS81 recruitment relies on chromatin modifier EZH2. Prevention of MRE11-mediated fork degradation requires RAD51, which is facilitated by HR-Fanconi anemia proteins but antagonized by RADX. Thus, RAD51 both promotes and prevents fork degradation, in particular, at the steps of fork reversal and through the formation of stabilized filaments, respectively. Prevention of DNA2-mediated fork degradation involves BOD1L and ABRO1. Proteins that contribute to fork protection and degradation are labeled with a red and green color, respectively. Asterisk, MUS81’s role in fork degradation is not always observed (discussed in the text). (B) Summary of the reported genetic interactions between FP proteins and proteins that directly or indirectly promote fork degradation, simplified here as “fork degradation proteins”. A checkmark indicates that the absence of a FP protein leads to nascent strand degradation involving the corresponding fork degradation protein. An x indicates that evidence exists that a given fork degradation protein is not responsible for fork degradation in the absence of the corresponding FP protein. (x) indicates that EXO1 was not tested for BOD1L. Citations of proteins involved in fork protection and degradation are listed.

Figure 4.. Separation-of-function systems to study HR and FP functions.

Three separation-of-function systems to dissect the individual contributions of HR and FP pathways have been described (Feng and Jasin 2017).

Figure 5.. Context-dependent requirement for HR versus FP for cell survival.

Differential requirements for HR and FP are observed to reach the threshold of cell viability in different contexts of BRCA2 deficiency, as highlighted in the dashed blue boxes. BRCA2 loss alone (untreated or with chemotherapy) (red arrows) is shown as a baseline for comparison to BRCA2 loss with the indicated additional genetic alterations (black arrows). (A) Nontransformed mammary epithelial cells MCF10A do not survive BRCA2 loss. Restoration of HR, but not FP, re-establishes cell viability. p53 loss fails to suppress apoptosis and only partially rescues cell proliferation. (B) In mouse ES cells, FP restoration is sufficient to rescue cell survival after BRCA2 loss. These cells may have a higher tolerance to DNA damage than MCF10A cells due to a compromised p53 response. (C) In BRCA2-deficient tumor cells, FP is also sufficient to overcome the viability threshold to confer chemoresistance. Presumably, these cells have evolved to survive BRCA2 loss and aberrantly proliferate (purple face) and thus may have a higher tolerance to DNA damage.