Abstract
Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are <or=0.3 to <or=2.8 x 10-4. BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase beta is replaced by DNA polymerase beta variants that fill single nucleotide gaps with lower fidelity. Under these conditions, the base substitution fidelity of polymerase beta-dependent BER is 3-8-fold higher than is single nucleotide gap filling by polymerase beta alone. Thus other proteins in the BER reaction may enhance the base substitution fidelity of DNA polymerase beta during single nucleotide BER.
Highlights
Damaged DNA bases are removed from mammalian erization, the displaced dRP group is excised, and the resulting genomes by base excision repair (BER)
BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase  is replaced by DNA polymerase  variants that fill single nucleotide gaps with lower fidelity
The results establish the minimum fidelity of this single nucleotide BER reaction and indicate that BER fidelity is at least severalfold higher than is the fidelity of single nucleotide gap filling DNA synthesis by pol  alone
Summary
Vol 278, No 28, Issue of July 11, pp. 25947–25951, 2003 Printed in U.S.A. The Base Substitution Fidelity of DNA Polymerase -dependent Single Nucleotide Base Excision Repair*. Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5,2-deoxyribose-5-phosphate lyase Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000fold. Three other human DNA polymerases have dRP lyase activity, pol ␥ [5], pol [6], and pol [7], and pol and pol can substitute for pol  in BER reactions in vitro that repair uracil paired opposite adenine or guanine [6, 7] Such enzymatic redundancy is interesting because the base substitution fidelity of these four DNA polymerases differs over a remarkable range when they work alone to fill gaps in DNA. We measure base substitution error rates for repair of uracil opposite adenine or guanine, in single nucleotide BER reactions reconstituted with purified proteins, one of which is human pol . This paper is available on line at http://www.jbc.org
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
