Moving Mountains-The BRCA1 Promotion of DNA Resection

Abstract

DNA double-strand breaks (DSBs) occur in our cells in the context of chromatin. This type of lesion is toxic, entirely preventing genome continuity and causing cell death or terminal arrest. Several repair mechanisms can act on DNA surrounding a DSB, only some of which carry a low risk of mutation, so that which repair process is utilized is critical to the stability of genetic material of cells. A key component of repair outcome is the degree of DNA resection directed to either side of the break site. This in turn determines the subsequent forms of repair in which DNA homology plays a part. Here we will focus on chromatin and chromatin-bound complexes which constitute the "mountains" that block resection, with a particular focus on how the breast and ovarian cancer predisposition protein-1 (BRCA1) contributes to repair outcomes through overcoming these blocks.

Keywords: 53BP1; BRCA1; SMARCAD1; USP48; homologous recombination; resection.

Figure 1

A diagram to show how resection influences repair pathway choice. Approximately 80% of DSBs are repaired by classical NHEJ which does not require resection. aNHEJ or MMEJ requires minimal resection to expose regions of microhomology. Long range resection is required for the major HDR pathways gene conversion (GC) and single-strand annealing (SSA). Key proteins in each pathway are given in red.

Figure 2

Following a DSB 53BP1 interacts with modified histones, (H2A-K15-ubiquitin blue circles, H4K20-dimethylation, green hexagons), the 53BP1-RIF1-Shieldin (Shld1-Shld2-Shld3-Rev7-CST) complex is recruited to sites of DNA damage where it also prevents retention of BRCA1-BARD1. Shld2 binds directly to ssDNA stretches >50 nucleotides long via three OB-folds. Together the Shieldin complex recruits DNA Polα which in turn primes DNA synthesis to fill in resected DNA ends. This prevents long range resection and repair by HDR pathways and supports repair by NHEJ.

Figure 3

Chromatin compaction around a single DSB changes with time. Immediately following damage (within 10 min) local compaction occurs which has been linked to transcriptional repression, limiting movement of the break ends, and to strip and prime chromatin modifications for repair. At 30–60 min post repair chromatin density is at its greatest beyond the 53BP1 boundary that marks the break site. In S/G2, the 53BP1 boundary is repositioned by BRCA1-BARD1 to open up the damage site for long range resection. In addition the 53BP1 damage complexes are thought to have liquid like phase properties which may be key to these large scale (5 μm diameter) effects on chromatin densities.

Figure 4

Multiple mechanisms contribute to SMARCAD1 recruitment to DNA damage sites. BRCA1-BARD1 modification of H2A-K125/127/129ub is recognized by SMARCAD1 CUE domains. Phosphorylation events by ATM (SMARCAD1-T906) facilitate recruitment and CDKs (SMARCAD1-T71) promote TOPBP1 interaction. SMARCAD1 preferentially binds and is activated by ssDNA-nucleosomes. Finally, SMARCAD1 has been proposed to directly bind H3K27Ac and H3R26cit although the role of these interactions in the DDR has yet to be characterized.

Figure 5

A new BRCA1-circuit that controls DNA repair pathway choice. In S-phase BRCA1-BARD1 is retained at DNA double strand break sites where it mono-ubiquitinates the extreme C-terminus of H2A at K125/127/129 (1). This ubiquitination modification is recognized by the chromatin remodeler SMARCAD1 (2) which remodels nucleosomes to promote 53BP1 repositioning at the break site (53BP1 binds modified H2A-K15-ubiquitin blue circles, H4K20-dimethylation, green hexagons). This allows recruitment of long-range resection enzymes, such as DNA2, BLM or EXO1, required for homology-directed repair. The deubiquitinating enzyme USP48 specifically removes the BRCA1-mediated H2A-Ub modification (3) to prevent over-resection and limit use of the mutagenic single-strand annealing repair pathway.