Proteomic analysis of DNA synthesis on a structured DNA template in human cellular extracts: Interplay between NHEJ and replication-associated proteins

ABSTRACT: It has been established that short inverted repeats (SIRs) trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with this structured DNA is unknown. We have previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded DNA template is transiently blocked at DNA hairpins [1](Schmutz et al., 2007). Here, we report the proteomic analysis of proteins bound to the DNA template providing evidence that proteins of the NHEJ pathway, particularly the DNA-PK complex (composed of DNA-PKcs and the Ku70/Ku80 dimer) recognize structured single-stranded DNA. DNA-PKcs inhibition results in the mobilization on the template DNA of the DNA-PK complex, along with other proteins acting downstream in the NHEJ pathway, especially the XRCC4-DNA Ligase 4 complex and the recently identified cofactor PAXX. The retention of NHEJ factors to the template DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that NHEJ proteins may hinder the progression of the DNA synthesis at these sites. Conversely, hijacking of the DNA-PK complex by double-stranded oligos (dsO) results in a large removal of the pausing sites and an elevated DNA extension efficiency. Overall these results raise the possibility that, upon binding to DNA hairpins formed onto ssDNA during fork progression, the DNA-PK complex may play some role in replication fork dynamics in vivo, role that is however not related to repair of double-strand breaks.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Lauriane Kuhn  

LAB HEAD: Régine Janel

PROVIDER: PXD011156 | Pride | 2020-02-04

REPOSITORIES: Pride