Nucleic Acids Research, 2001, Vol. 29, No. 15 3277-3288
© 2001 Oxford University Press
The role of DNA polymerase activity in human non-homologous end joining
1Biocenter Oulu and Department of Biochemistry, PO Box 3000, FIN-90014 University of Oulu, Finland and 2Department of Biology, PO Box 101, University of Joensuu, FIN-80101 Joensuu, Finland
In mammalian cells, DNA double-strand breaks are repaired mainly by non-homologous end joining, which modifies and ligates two DNA ends without requiring extensive base pairing interactions for alignment. We investigated the role of DNA polymerases in DNA-PK-dependent end joining of restriction-digested plasmids in vitro and in vivo. Rejoining of DNA blunt ends as well as those with partially complementary 5' or 3' overhangs was stimulated by 20–53% in HeLa cell-free extracts when dNTPs were included, indicating that part of the end joining is dependent on DNA synthesis. This DNA synthesis-dependent end joining was sensitive to aphidicolin, an inhibitor of 
-like DNA polymerases. Furthermore, antibodies that neutralize the activity of DNA polymerase were found to strongly inhibit end joining in vitro, whereas neutralizing antibodies directed against DNA polymerases ß and 
did not. DNA sequence analysis of end joining products revealed two prominent modes of repair, one of which appeared to be dependent on DNA synthesis. Identical products of end joining were recovered from HeLa cells after transfection with one of the model substrates, suggesting that the same end joining mechanisms also operate in vivo. Fractionation of cell extracts to separate PCNA as well as depletion of cell extracts for PCNA resulted in a moderate but significant reduction in end joining activity, suggesting a potential role in a minor repair pathway.
* To whom correspondence should be addressed at: Biocenter Oulu and Department of Biochemistry, PO Box 3000, FIN-90014 University of Oulu, Finland. Tel: +358 8 553 1155; Fax: +358 8 553 1141; Email: juhani.syvaoja{at}oulu.fi The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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