Accurate in Vitro End Joining of a DNA Double Strand Break with Partially Cohesive 3′-Overhangs and 3′-Phosphoglycolate Termini

Abstract

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3-overhangs and a onebase gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3 3 5 resection before ligation, was formed. Similar results were seen for a substrate with 5-overhangs and recessed 3-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

Regulation and mechanisms of mammalian double-strand break repair

“…These and other results have led to the hypothesis that DNA-PKcs sequesters DNA ends, protecting them from degradation until an end-joining partner is found. At this point, the two end-bound DNA-PKcs molecules may synapse, phosphorylate each other and dissociate (or at least relinquish their position at the extreme end of DNA), thereby allowing processing of the aligned ends to proceed (Chu, 1997;Calsou et al, 1999;Karran, 2000;Chen S et al, 2001). Recent kinetic data suggesting that such synapsis is required for DNA-PKcs activation lend further support to such a model (DeFazio et al, 2002).…”

“…Additional candidates for participation in NHEJ include Artemis , the MRE11/ RAD50/NBS1 MRN complex (Huang and Dynan, 2002), BRCA1 (Zhong et al, 2002), tyrosyl-DNA phosphodiesterase (TDP1) (Inamdar et al, 2002) and APE1 (Demple and Harrison, 1994;Suh et al, 1997). Much of the DNA processing associated with NHEJ was first described in Xenopus laevis egg extracts (Thode et al, 1990;Gu et al, 1996Gu et al, , 1998Labhart, 1999), but nearly all the findings from this system have proven valid in the more recently developed mammalian systems (Baumann and West, 1998;Feldmann et al, 2000;Chen S et al, 2001).…”

“…A diverse body of evidence from studies with cell-free extracts and purified proteins implicates both KU and XRCC4/ligase IV in the alignment of partially cohesive ends for gap filling (Cary et al, 1997;Pang et al, 1997;Yaneva et al, 1997;Chen et al, 2000;Feldmann et al, 2000;Chen S et al, 2001;Lee et al, 2003). The gapfilling polymerase is most likely either DNA polymerase m (POLm or POL l).…”

“…Nevertheless, rodent and, in some cases, human cells lacking the proteins required for NHEJ are viable -a fact that has greatly facilitated both the cloning of the genes encoding these factors and elucidation of their specific roles in the end-joining process. Known NHEJ proteins include KU, DNA-PKcs, DNA ligase IV, its cofactor XRCC4 (Chu, 1997;Calsou et al, 1999;Karran, 2000;Chen S et al, 2001), and probably DNA polymerase m and/or (Mahajan et al, 2002) and polynucleotide kinase/phosphatase (PNKP) (Chappell et al, 2002). Additional candidates for participation in NHEJ include Artemis , the MRE11/ RAD50/NBS1 MRN complex (Huang and Dynan, 2002), BRCA1 (Zhong et al, 2002), tyrosyl-DNA phosphodiesterase (TDP1) (Inamdar et al, 2002) and APE1 (Demple and Harrison, 1994;Suh et al, 1997).…”

Chromatin dynamics coupled to DNA repair

“…72 After alignment of these complementary sequences, the remaining DNA is removed by a nuclease and the gaps are filled by a DNA polymerase. This repair process is mediated by the Ku70/Ku80 heterodimer, which binds the broken DNA ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme.…”