BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy

The early notion that cancer is caused by mutations in genes critical for the control of cell growth implied that genome stability is important for preventing oncogenesis. During the past decade, knowledge about the mechanisms by which genes erode and the molecular machinery designed to counteract this time-dependent genetic degeneration has increased markedly. At the same time, it has become apparent that inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer. This review summarizes the main DNA caretaking systems and their impact on genome stability and carcinogenesis.

Genome maintenance mechanisms for preventing cancer

The DNA Damage Response: Making It Safe to Play with Knives

https://www.molmed.nl/uploads/abstracts/1102/Gent_van1_olaparib%20BrCa%202010%20lancet.pdf

Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial

Despite the skepticism that once prevailed among immunologists, it is now widely accepted that the normal immune system harbors a T-cell population, called regulatory T cells (Treg cells), specialized for immune suppression. It was first shown that depletion of a T-cell subpopulation from normal rodents produced autoimmune disease. Search for a molecular marker specific for such autoimmune-preventive Treg cells has revealed that the majority, if not all, of them constitutively express the CD25 molecule as depletion of CD25+CD4+ T cells spontaneously evokes autoimmune disease in otherwise normal rodents. The expression of CD25 by Treg cells has made it possible to delineate their developmental pathways, in particular their thymic development, and establish simple in vitro assay for assessing their suppressive activity. The marker and the in vitro assay have helped to identify human Treg cells with similar functional and phenotypic characteristics. Recent efforts have shown that natural Treg cells specifically express the transcription factor Foxp3 and that mutations of the Foxp3 gene produce a variety of immunological diseases in humans and rodents. Specific expression of Foxp3 in natural Treg cells has enabled their functional and developmental characterization by genetic approach. These studies altogether have provided firm evidence for Foxp3+CD25+CD4+ Treg cells as an indispensable cellular constituent of the normal immune system for establishing and maintaining immunologic self-tolerance and immune homeostasis. Treg cells are now within the scope of clinical use to treat immunological diseases and control physiological and pathological immune responses.

Regulatory T Cells

Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

/pdf/xrcc3-promotes-homology-directed-repair-of-dna-damage-in-35plya38u1.pdf

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells

https://www.cell.com/molecular-cell/pdf/S1097-2765(01)00174-5.pdf

BRCA2 is required for homology-directed repair of chromosomal breaks.

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70−/−, XRCC4−/−, and DNA-PKcs−/− cells, with the increase being particularly striking in Ku70−/− cells. Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins. A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.

/pdf/ku-dna-end-binding-protein-modulates-homologous-repair-of-4qqehaw0fg.pdf

Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 / mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential

/pdf/genetic-steps-of-mammalian-homologous-repair-with-distinct-1cfmen03jh.pdf

Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca–/– cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

/pdf/human-fanconi-anemia-monoubiquitination-pathway-promotes-w2n4ruiwwv.pdf

Andrew J. Pierce

Papers

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells

BRCA2 is required for homology-directed repair of chromosomal breaks.

Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells

Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair