NMR studies on the structure of the DNA binding domain A of replication protein A from S. cerevisiae and interactions of RPA homologues with single stranded DNA

Abstract

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single stranded (ss) DNA binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA-XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, NMR spectroscopy was used to assess the binding of the tandem high affinity DNA binding domains, RPA70AB, and of the isolated domain RPA70A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA70A and RPA70AB were found to bind nonspecifically to both strands of normal and CPD-containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA binding domain of XPA(XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA70A and RPA70AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER. Replication Protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA--T antigen (T-ag) interaction required for Simian Virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate th...