Ribonucleotides as nucleotide excision repair substrates

Abstract

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10 to 20 fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (A. Vaisman, J.P. McDonald, D. Huston, W, Kuban, L. Liu, B. Van Houten, and R. Woodgate, PLoS Genetics, in press, 2013) reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide--with its unique electrostatic and hydrogen bonding properties--may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.

Processing ribonucleotides incorporated during eukaryotic DNA replication

“…Nucleotide excision repair (NER), a multistep DNA repair process that excises bulky ultraviolet- (UV) or chemical-induced lesions, has been implicated to act as a backup pathway for ribonucleotide removal in RER-deficient bacterial cells 63,64 . However, ribonucleotides in DNA are a poor substrate for the human NER system in vitro and ribonucleotides are not efficiently removed by NER in human cells 65 .…”

Redundancy in ribonucleotide excision repair: Competition, compensation, and cooperation

“…Furthermore, despite its small impact on the global helical properties of the DNA duplex, an isolated rNMP can be replaced as efficiently as various bulky DNA-distorting lesions. Based on the results of molecular dynamics simulations, Cai et al ., [52] have proposed that ribonucleotide recognition by the UvrAB relies on hydrogen bonding and electrostatic interactions of the ribose 2'-OH group with several aromatic residues in UvrB that are responsible for damage recognition.…”

Ribonucleotides in bacterial DNA

“…In addition to the RNase H system employed for the removal of RNA from genomic DNA, topo I removes single rNMPs in S. cerevisiae and nucleotide excision repair (NER) can remove both single rNMPs and stretches of rNMPs in E. coli (Kim et al, 2011; Vaisman et al, 2013; Cai et al, 2014). In this section, we review the processes impacting removal of rNMPs incorporated into genomic DNA.…”

“…In yeast, topoisomerase I removes single embedded rNMPs yielding a 2–5 bp deletion, which can be mitigated by helicase Srs2 in collaboration with Exo I (Kim et al, 2011; Sekiguchi and Shuman, 1997; Williams et al, 2013; Potenski et al, 2014). Woodgate and co-workers again used the frequent rNMP incorporation by UmuCY11A variant and found that nucleotide excision repair (NER) serves as a backup pathway for removal of rNMPs in cells deficient for rnhB [(Vaisman et al, 2013) for review (Cai et al, 2014)].…”

“…How does NER recognize single rNMPs in DNA? A recent model has been proposed where the 2′-OH in the ribose sugar forms electrostatic and hydrogen bonding interactions with tyrosine residues in the β-hairpin of UvrB resulting in “damage” recognition (Cai et al, 2014). Although this model needs to be tested biochemically, it provides a basis for understanding ribose sugar recognition by NER.…”