DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining.

Abstract:

:Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 hetero-dimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Feldmann E,Schmiemann V,Goedecke W,Reichenberger S,Pfeiffer P

doi

10.1093/nar/28.13.2585

keywords:

subject

Has Abstract

pub_date

2000-07-01 00:00:00

pages

2585-96

issue

13

eissn

0305-1048

issn

1362-4962

journal_volume

28

pub_type

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