RECOGNITION AND REPAIR OF MISMATCHED BASE-PAIRS IN DNA

Laurence Pearl

G:U and G:T mismatched base-pairs can arise in DNA by the spontaneous deamination of cytosine and 5-methylcytosine respectively, and by misincorporation during DNA replication. If not recognised and repaired prior to the next round of replication, these mismatched base-pairs generate G:C -> A:T transition mutations in half the progeny cells.

A base-excision repair pathway for uracil has been identified in prokaryotes and eukaryotes, initiated by uracil-DNA glycosylase (UDG). This highly conserved enzyme hydrolyses the N-glycosidic bond connecting the uracil base to the sugar, generating an abasic site which is the substrate for subsequent enzymes in the pathway. UDGs are exquisitely specific for the excision of only uracil, but from single or double stranded DNA, regardless of context. The structural and mechanistic basis for this specificity will be described.

An ability to recognise and repair uracil in DNA, allows organisms to combat this major inherent source of genomic instability, but necessitates the evolution of a tagged-uracil (thymine) as a base-pairing partner for adenine in DNA. The DNA- bacteriophage PBS1 is unique in utilising uracil instead of thymine in its DNA. The biological adaptations required by this strategy will be discussed, and the structural basis for PBS1s necessary resistance to UDG activity will be described.

The recognition of an unnatural base such as uracil in DNA, can be achieved by UDG without consideration of the context in which it occurs. Recognition of a mismatch such as G:T, presents a more complex challenge, as both bases are natural to DNA, and it is only their mismatch that is unusual. Mismatch-specific DNA glycosylases have recently been described (MUG in bacteria, TDG in eukaryotes), which will excise both uracil and thymine bases, but only from mismatches with guanine in double-stranded DNA. Despite no detectable amino acid sequence homology, structural studies of the bacterial MUG enzyme reveal a remarkable structural similarity to the UDG enzymes, despite no significant sequence homology. Structural and mechanistic studies of this new family of enzymes will be described.

Although uracil and 5-methyl uracil deamination are a universal problem for DNA- organisms, so far no homologues of the UDG or MUG/TDG families of enzymes have been identified in the third kingdom, the Archaea. Nontheless these organisms clearly display an ability to maintain the integrity of their DNA. Preliminary data identifying a novel uracil detection and repair pathway will be presented.