In vitro analysis of the zinc-finger motif in human replication protein A

Jiaowang Dong, Jang Su Park, Suk-Hee Lee

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes:replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase α-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys→Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase S activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.

Original languageEnglish
Pages (from-to)311-317
Number of pages7
JournalBiochemical Journal
Volume337
Issue number2
DOIs
StatePublished - Jan 15 1999

Fingerprint

Zinc Fingers
Replication Protein A
Zinc
Repair
Single-Stranded DNA
Mutant Proteins
DNA-Directed DNA Polymerase
DNA Repair
DNA
Nucleotides
DNA-Activated Protein Kinase
Recombinational DNA Repair
Phosphorylation
Cyclin-Dependent Kinases
In Vitro Techniques
human RPA1 protein
Eukaryota
DNA Replication
Metabolism
Cysteine

Keywords

  • DNA repair
  • DNA replication
  • Single-stranded DNA binding
  • Xeroderma pigmentosum group A complementing protein

ASJC Scopus subject areas

  • Biochemistry

Cite this

In vitro analysis of the zinc-finger motif in human replication protein A. / Dong, Jiaowang; Park, Jang Su; Lee, Suk-Hee.

In: Biochemical Journal, Vol. 337, No. 2, 15.01.1999, p. 311-317.

Research output: Contribution to journalArticle

Dong, Jiaowang ; Park, Jang Su ; Lee, Suk-Hee. / In vitro analysis of the zinc-finger motif in human replication protein A. In: Biochemical Journal. 1999 ; Vol. 337, No. 2. pp. 311-317.
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