Structure and function in the uracil-DNA glycosylase superfamily

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Abstract

Deamination of cytosine to uracil is one of the major pro-mutagenic events in DNA, causing G:C→A:T transition mutations if not repaired before replication. Repair of uracil-DNA is achieved in a base-excision pathway initiated by a uracil-DNA glycosylase (UDG) enzyme of which four families have so far been identified. Family-1 enzymes are active against uracil in ssDNA and dsDNA, and recognise uracil explicitly in an extrahelical conformation via a combination of protein and bound-water interactions. Extrahelical recognition requires an efficient process of substrate location by ‘base-sampling’ probably by hopping or gliding along the DNA. Family-2 enzymes are mismatch specific and explicitly recognise the widowed guanine on the complementary strand rather than the extrahelical scissile pyrimidine. This allows a broader specificity so that some Family-2 enzymes can excise uracil and 3,N4-ethenocytosine from mismatches with guanine. Although structures are not yet available for Family-3 (SMUG) and Family-4 enzymes, sequence analysis suggests similar overall folds, and identifies common active site motifs but with a surprising lack of conservation of catalytic residues between members of the super-family.

Introduction

Deamination of the normal DNA base cytosine to form uracil, represents one of the major underlying instabilities in the encoding of genetic information in DNA. The conversion of cytosine, the base-pairing partner of guanine, into uracil, a fully competent base-pairing partner for adenine, causes a G:C→A:T transition mutation in half of the progeny on replication (Fig. 1). Although the inherent rate of this reaction is low, a typical human cell with a genome of 1010 base pairs, would expect to experience several hundred such changes per day, with potentially disastrous changes to the coding sequences of its proteins. Unlike many of the other species formed by unwanted chemical modification of normal DNA bases, uracil is a normal cellular component and is used as the base-pairing partner to adenine in RNA. However, the possibility of its formation by cytosine deamination makes uracil itself unsuited for this role in the long-term informational storage required of DNA. Instead, adenine in DNA is base-paired to 5-methyl-uracil (better known as thymine) which is used for no other purpose than template-directed DNA synthesis, and can be simply distinguished from pro-mutagenic uracil arising by deamination of cytosine. The use of a distinct ‘tagged’ uracil analogue for intentional incorporation opposite adenine, permits specific repair of unintentional uracil generated by cytosine deamination, and puts a brake on the inexorable transition from C:G→A:T/U that would otherwise occur.

The first observation of an activity involved in repair of uracil in DNA, was made by Lindahl [1]. This enzyme purified from Escherichia coli, uracil-DNA glycosylase (UDG), hydrolysed the N-glycosidic bond connecting the base to the deoxyribose sugar of the DNA backbone, releasing free uracil base and DNA containing an abasic site, as its products. In subsequent steps, the abasic sugar and its 3′-phosphate are removed, a template-directed DNA polymerase inserts the missing nucleotide, and the backbone is closed by DNA ligase, in a generic base-excision repair pathway. In principle, cytosine deamination could be directly reversed by re-amination of the uracil, a chemically trivial reaction, and indeed, how cytosine base is biologically synthesised in the first place. However, this would be highly mutagenic, as some uracil becomes incorporated in A:U base pairs in DNA during replication due to the inability of DNA polymerases to distinguish TTP from dUTP in template-directed DNA synthesis. A direct re-amination pathway that did not take account of the base-pairing context of the uracil, would correct pro-mutagenic G:U formed by deamination, but convert innocuous A:U base-pairs to mutagenic A:C mispairs. The base-excision pathway, which utilises the information contained in the second strand, is able to convert both G:U and A:U base-pairs back to their correct G:C and A:T Watson–Crick configurations using the same set of reactions, and appears, so far, to be the universal mechanism for uracil repair.

Section snippets

Substrate recognition by Family-1 UDG

The archetypal UDGs, homologous to that first described in E. coli, (henceforth Family-1) are able to excise uracil base efficiently from single-stranded DNA, and from double-stranded DNA regardless of the partner base on the second strand. They are exquisitely specific for uracil in DNA, and show negligible activity towards the natural DNA bases cytosine or thymine, or uracil in RNA. The structural basis for this specificity is now well understood as a result of structural studies of ligand

Substrate recognition by MUG/TDG enzymes

Until relatively recently, and apart from very low and probably spurious activities displayed by proteins with other main functions [11], [12], all authentic uracil DNA–glycosylases have been homologues of the archetypal E. coli enzyme. These are characterised by activity against uracil in ssDNA or dsDNA, susceptibility to inhibition by the phage PBS1/2 UGI protein [13], and strong conservation of the key residues implicated in specificity and catalysis [2], [3]. An N-glycosylase activity

Eukaryotic SMUG1–ssDNA selective UDG

In the past year, two new UDG activities have been identified. The first of these came as a result of screening eukaryotic expression libraries for genes encoding proteins with affinity for oligonucleotides incorporating base-excision repair transition state analogues [28]. A gene encoding a 31-kDa protein with high-affinity for these oligonucleotides was isolated from a Xenopus library and a close homologue identified in human ESTs. As the transition state analogues utilised in the binding

Conclusion

The ability to repair deaminated cytosine and thereby minimise one of the major informational instabilities of DNA, provides an important selective advantage to a complex organism, and is likely to have arisen very early in cellular evolution. UDG activities corresponding to one family or other have been identified in organisms from almost all kingdoms, including the archaea, and in the Pox and Herpes viruses. Several organisms have multiple examples, so that humans and other higher eukaryotes

Acknowledgements

I am very grateful to Renos Savva, Tracey Barrett, Mark Roe, Bernard O'Hara and Martin Greagg for their contributions to studies of uracil repair in my laboratory, and to Josef Jiricny, Greg Verdine, Bernard Connolly, George Panayotou and Tom Brown for valued collaborations. Studies of DNA Repair in my laboratory are funded by the Cancer Research Campaign and the UK Biotechnology and Biological Sciences Research Council.

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