An Affinity of Human Replication Protein A for Ultraviolet-damaged DNA
IMPLICATIONS FOR DAMAGE RECOGNITION IN NUCLEOTIDE EXCISION REPAIR (*)
- From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
- § To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8601; Fax: 409-747-8608; lprakash{at}scms.utmb.edu.
Abstract
Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication.
Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for
single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision
of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment
preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation
of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg
in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property
of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.
Footnotes
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↵* This work was supported by Grant DE-FGO3-93ER61706 from the Department of Energy and Grant CA41261 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- NER
-
nucleotide excision repair
- RPA
-
replication protein A
- ss
-
single-stranded
- DTT
-
dithiothreitol
- BSA
-
bovine serum albumin
- CPD
-
cyclobutane pyrimidine dimer.
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- Received February 5, 1996.
- Revision received March 12, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
