Implication of DNA Polymerase λ in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts

Abstract

Accurate repair of free radical-mediated DNA doublestrand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1-or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases and , both of which were present in the extracts. For a DNA substrate with partially complementary 3 overhangs and a 2-base gap, antibodies to polymerase completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase had little effect. Immunodepletion of polymerase , but not polymerase , likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase . Recombinant polymerase , and a truncated polymerase lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase ␤ was completely inactive. The results suggest that polymerase is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.

DNA polymerases in adaptive immunity

“…Polµ and Polλ have been shown to associate with the protein complex Ku-DNA-PKcs once they are bound to DNA and can perform gap-filling synthesis of paired DNA ends in vitro 11,12,18 . The possible use of these enzymes during NHEJ seems to be due to their capacity to synthesize DNA from less stable primer structures compared to replicative polymerases, such as primers that encompass a DNA template with a gap 19 (Fig.3c).…”

Role of the catalytic metal during polymerization by DNA polymerase lambda

“…This flexibility was first suggested by the observation that Pol λ generates deletion errors at an extremely high rate [11], and is now thought to be a feature of the enzyme that facilitates its role in vivo. The phenotypes of mice deficient in this polymerase indicate that Pol λ plays a role in the V(D)J process of antigen gene diversification [12], and several reports implicate Pol λ in Base Excision Repair [13][14][15] and the repair of double-strand breaks through the Non-Homologous DNA End-Joining pathway [16][17][18]. It is thus interesting to examine the details of the polymerization reaction as catalyzed by Pol λ in order to understand whether specific catalytic features can account for these special properties.…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…As with other BRCT domains, the BRCT domain in TdT can also interact with a phosphoserine-containing motif, but whether this has functional relevance is not yet clear [Yu et al, 2003]. Rather, in all examples tested (Pol4 [Tseng and Tomkinson, 2002] as well as vertebrate Pol k [Fan and Wu, 2004;Lee et al, 2004;Ma et al, 2004;Nick McElhinny et al, 2005;Mueller et al, 2008], Pol l [Mahajan et al, 2002;Ma et al, 2004;Nick McElhinny et al, 2005;DeRose et al, 2007] and TdT), the BRCT domain is required for physical interaction with Ku and XRCC4-ligase IV at DNA ends, and there is as yet Environmental and Molecular Mutagenesis. DOI 10.1002/em no contribution of Ku, XRCC4, or ligase IV phosphorylation.…”

“…Pol k was first clearly linked to NHEJ by Dr. Povirk's group, who demonstrated immunodepletion of Pol k in HeLa cell extracts blocked the ability of NHEJ to resolve ends with 3 0 overhangs that could be aligned to generate a short one-nucleotide gap [Lee et al, 2004]. Pol k was later shown to promote NHEJ in reactions reconstituted Environmental and Molecular Mutagenesis.…”