Maureen Biggerstaff

Mammalian DNA nucleotide excision repair reconstituted with purified protein components

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer‐prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP‐A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP‐A DNA repair protein by complementation of cell extracts from a repair‐defective human XP‐A cell line. XP‐A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP‐A protein binds preferentially to ultraviolet light‐irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP‐A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP‐A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins.

Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitro.

The mammalian ERCC1‐encoded polypeptide is required for nucleotide excision repair of damaged DNA and is homologous to Saccharomyces cerevisiae RAD10, which functions in repair and mitotic intrachromosomal recombination. Rodent cells representing repair complementation group 1 have nonfunctional ERCC1. We report that repair of UV‐irradiated DNA can be reconstituted by combining rodent group 1 cell extracts with correcting protein from HeLa cells. Background repair was minimized by employing fractionated rodent cell extracts supplemented with human replication proteins RPA and PCNA. Group 1‐correcting activity has a native molecular mass of 100 kDa and contains the 33 kDa ERCC1 polypeptide, as well as complementing activities for extracts from rodent group 4 and xeroderma pigmentosum group F (XP‐F) cells. Extracts of group 1, group 4 or XP‐F cells do not complement one another in vitro, although they complement extracts from other groups. The amount of ERCC1 detectable by immunoblotting is reduced in group 1, group 4 and XP‐F extracts. Recombinant ERCC1 from Escherichia coli only weakly corrected the group 1 defect. The data suggest that ERCC1 is part of a functional protein complex with group 4 and XP‐F correcting activities. The latter two may be equivalent to one another and analogous to S. cerevisiae RAD1.

Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesis

Extracts from HeLa cells were used to study the susceptibility of repair synthesis in UV-irradiated plasmid DNA to inhibition by exogenously added nucleic acid. Purified DNA restriction fragments have little inhibitory effect on repair synthesis. However, activated calf thymus DNA fragments, genomic DNA fragments in cell extracts, and sonicated plasmid DNA all inhibited repair synthesis. Degraded DNA fragments arising from E. coli during bacterial plasmid purification were found to be particularly inhibitory. tRNA is not a potent inhibitor of in vitro repair synthesis. In order to observe efficient DNA repair synthesis mediated by human cell extracts, it is essential to prepare highly purified closed circular plasmid DNA, and we describe a reliable method for doing so.

Repair Synthesis Assay for Nucleotide Excision Repair Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA

Methods are described for measuring nucleotide excision repair (NER) of damaged plasmid DNA using fractionated mammalian cell extracts. NER creates a single-stranded gap of approx 25-30 nt. Filling of this gap by repair synthesis can be monitored by the incorporation of radioactive nucleotides. We first describe the preparation of ultraviolet light (UV)-damaged and control plasmid DNA substrates and purification of their closed-circular forms. To increase the specificity for NER, plasmid molecules containing pyrimidine hydrates and other lesions sensitive to Escherichia coli Nth protein are eliminated. The preparation of whole cell extracts active in NER is described, both for cells grown as attached cultures and those grown in suspension. Cell extracts are partially purified on phosphocellulose to produce a fraction that can carry out the full NER reaction when combined with purified RPA and PCNA proteins. This enables NER to be quantified in an assay with exceptionally low background in nondamaged DNA.