To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection

Abstract

The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.

Keywords: DNA repair; double-strand break; homologous recombination; non-homologous end joining; telomeres.

FIGURE 1

Overview of DSB repair pathways. Left, homologous recombination (HR) involves resection of the DNA ends by various nucleases. The ssDNA generated is then bound by RPA. Next, there is an exchange of RPA for RAD51, which facilitates the homology search and repair of the DSB. Center, for non-homologous end joining (NHEJ), the DNA ends are bound by Ku70/80 heterodimers promoting the binding of DNA-PKcs. This creates a binding platform for XRCC4, XLF, and LIG4, which facilitate ligation of the DNA ends. Right, DSBs can also be repaired by alternative-end joining (alt-EJ) or single strand annealing (SSA) pathways. These involve the use of short homologous sequences that are exposed by resection of the break. Following alignment, the DNA flaps are removed and the DNA ligated. Alt-EJ uses around 2 to 20 base pairs (bp) of homology and SSA > 25 bp to align sequences.

FIGURE 2

Overview of telomeres. (A) Telomeres consist of a repetitive DNA sequence that forms a duplex DNA region and a 3′ G-rich ssDNA overhang (C-strand, red: G-strand, blue). (B) Telomeres are bound by the shelterin complex (TRF1-TRF2-RAP1-TIN2-TPP1-POT1) and the CST complex (CTC1-STN1-TEN1), which aid in telomere maintenance. (C) Steps in telomere replication. First, the telomere duplex is replicated resulting in either a blunt end (leading strand replication) or an overhang (lagging strand replication). The leading strand end is then processed to generate a G-overhang. Telomerase then extends the G-overhangs followed by C-strand fill-in to convert most of the ssDNA to duplex DNA, leaving a short G-overhang.

FIGURE 3

Key decision points in the repair of DSBs by HR. (1) The DNA ends are bound by either MRN or the Ku70/80 heterodimer. Binding and retention of MRN will shift repair toward HR and the binding of Ku shifts repair toward to NHEJ. (2) Short range resection by MRN. (3) Long-range resection of the DNA and RPA binding. (4) RPA is exchanged for RAD51, which facilitates strand invasion, DNA synthesis and HR repair.

FIGURE 4

How DSB ends are processed under different conditions. Scissors indicate MRN endonuclease activity. MRN binding to unblocked ends leads to resection of the DNA. When Ku binds in the absence of MRN, no resection occurs. However, when both Ku and MRN are bound, stimulation of the MRN nuclease activity promotes resection and the removal of Ku. MRN can similarly function at ends blocked by a protein adduct, damaged bases or DNA secondary structures.

FIGURE 5

TRF2 facilitates telomere loop (t-loop) formation by promoting invasion of the G-overhang into the duplex region to form a displacement loop (D-loop). This t-loop combined with shelterin blocks MRN and Ku from accessing the chromosome ends and initiating DSB repair.

FIGURE 6

Model of short-range resection. Short-range resection by MRN is stimulated by CtIP. Localization of CtIP to the chromatin is dependent on phosphorylation of CtIP by CDK and ATM and ubiquitination by BRCA1. CDK has also been proposed to phosphorylate MRX in budding yeast in a cell cycle-dependent manner, suggesting that CDK may regulate MRN in mammals. Phosphorylation of H2AX by ATM also promotes short-range resection and the recruitment of DSB machinery. Upon localization with MRN, CtIP stimulates both MRN endonuclease (indicated by the scissors) and 3′-to-5′ exonuclease activity to facilitate short-range resection. (P: phosphorylation; Ub: ubiquitination).

FIGURE 7

Model of telomere replication and end protection. During telomere replication, t-loops are resolved exposing chromosome ends to potential repair mechanisms. On the lagging strand, regions of ssDNA are bound by RPA as part of the normal replication process. To prevent stable RPA binding, an RPA-to-POT1 switch is facilitated by hnRNPA1. TERRA then removes hnRNPA1 to allow POT1 binding. On the leading strand, blunt telomere ends are resected by DNA2, EXO1 and/or Apollo to generate a G-overhang. (RPA may also bind to these ends and require removal). Telomerase is localized to the telomeres through its interaction with TPP1 to extend the G-overhang. To prevent G-overhang hyperextension, CST is localized to telomeres by TPP1 and inhibits telomerase activity. Additionally, CST promotes C-strand fill-in by stimulating pol α. After telomere processing, t-loops are reformed to protect the chromosome end from DSB repair.

FIGURE 8

Models of long-range resection and how long-range resection is inhibited/reversed. (A) Long-range resection is mediated by EXO1 or BLM/DNA2. The resulting ssDNA is bound by RPA, which serves as a platform for ATR binding. To inhibit NHEJ, TopBP1 bridges interactions between ATR and the BRCA1-BARD1 complex, blocking 53BP1. (B) 53BP1 is localized to DSBs through its interaction with specific histone marks. 53BP1 then interacts with RIF1, which localizes the shieldin complex. Shieldin inhibits long-range resection by preventing access to the resected DNA and reverses resection through its interaction with CST-pol α, which can mediate fill-in of the resected DNA. This promotes repair of the break by NHEJ. (P: phosphorylation; Ub: ubiquitination).

FIGURE 9

Potential mechanism for the protection of RPA-bound telomeres. Loss of POT1 or CST results in hyperextension of G-overhangs and telomeric RPA but very few fusions events. We propose that the combination of TRF2 and 53BP1 prevent fusions by blocking NHEJ and HR mediated repair, respectively.