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Recognition of damaged DNA by a replicative DNA polymerase

By Matt Wargo • June 3rd, 2010

Citation: Aller P, Ye Y, Wallace SS, Burrows CJ, Doublié S. 2010. Crystal structure of a replicative DNA polymerase bound to the oxidized guanine lesion guanidinohydantoin. Biochemistry 49, 2502-2509.

Direct link to the publication at the Biochemistry website

Authors’ Association with MMG:
Pierre Aller – is a postdoctoral associate in the MMG Department
Sylvie Doublié – is a Professor in the MMG Department
Susan S. Wallace – is Professor and Chair of the MMG Department
Prof. Cindy Burrows and Yu Ye are collaborators from the University of Utah.

The oxidation of guanine generates one of the most common DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). The further oxidation of 8-oxoG can produce either guanidinohydantoin (Gh) in duplex DNA or spiroiminodihydantoin (Sp) in nucleosides and ssDNA. Although Gh can be a strong block for replicative DNA polymerases such as RB69 DNA polymerase, this lesion is also mutagenic: DNA polymerases bypass Gh by preferentially incorporating a purine with a slight preference for adenine, which results in G.C –> T.A or G.C –> C.G transversions. The 2.15 A crystal structure of the replicative RB69 DNA polymerase in complex with DNA containing Gh reveals that Gh is extrahelical and rotated toward the major groove. In this conformation Gh is no longer in position to serve as a templating base for the incorporation of an incoming nucleotide. This work also constitutes the first crystallographic structure of Gh, which is stabilized in the R configuration in the two polymerase/DNA complexes present in the crystal asymmetric unit. In contrast to 8-oxoG, Gh is found in a high syn conformation in the DNA duplex and therefore presents the same hydrogen bond donor and acceptor pattern as thymine, which explains the propensity of DNA polymerases to incorporate a purine opposite Gh when bypass occurs.

Impact and Significance:
Replicative DNA polymerases copy genomic DNA with very high accuracy, making one error every 104-106 insertion events. If the enzyme harbors a proofreading activity (3’-5’ exonuclease) the replication accuracy is increased by an additional 10 to 100-fold. Our work focuses on the replicative DNA polymerase of the bacteriophage RB69, a structural homolog of human replicative polymerases. Our goal is to elucidate crystal structures of the RB69 polymerase bound to DNA containing an oxidized damaged base, in order to better understand the effect of the lesion on DNA replication. One of the most common and prevalent oxidative lesions found in DNA is 8-oxo-7,8-dihydroguanine (8-oxoG). Because of its very low oxidation potential, 8-oxoG can be further oxidized into two stable products: guanidinohydantoin (Gh), more commonly found in double stranded DNA, and spiroiminodihydantoin (Sp) more common in single stranded DNA.

This work represents the first crystallographic structure of Gh. The structural analysis also revealed why this lesion is a strong block to replication and allowed us to speculate why a purine is incorporated when bypass occurs.

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