Lesion selectivity in blockage of lambda exonuclease by DNA damage.

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites

The sequence specific interaction of ionising radiation, cisplatin and T4 endonuclease VII with DNA

Cisplatin and ionising radiation are mainstays of modern cancer treatment, and both are capable of damaging DNA. They act via different mechanisms; either the formation of DNA adducts or via the induction of DNA breaks. This thesis aims to examine the genomic location of these damage sites, at nucleotide resolution, as well as examine the inherent sequence specificity of T4 endonuclease VII cleavage without the presence of a structural substrate. A similar approach was used initially – linear amplification followed by fragment analysis to determine specificity in short DNA sequences. Secondly, next-generation sequencing methods were used to detect radiation-induced damage sites in the human genome, and an attempt was made to develop a genome-wide map of cisplatin-adduct formation. The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates, by cleaving DNA with staggered cuts. For the first time, the sequence specificity of cleavage sites was determined without the presence of a known DNA substrate. We found DNA was cleaved with a sequence specificity that conforms to AWTAA*STC, where A* indicates the cleavage site, W is A or T, and S is G or C. Cisplatin forms adducts with DNA, which cause distortions that prevent DNA replication. Adducts have been previously shown to form preferentially at GG nucleotides in short DNA sequences. It was shown that cisplatin adducts preferentially occurred at GG nucleotides in short DNA sequences derived from the mitochondrial genome; however, attempts to extrapolate this to a genome-wide scale were unsuccessful. Ionising radiation directly induces cytotoxic double-stranded DNA breaks, but is also subject to indirect attack from reactive oxygen species formed via radiolysis. Radiation-induced breaks are linear with respect to dose; however their precise location has not been determined. It was found that ionising radiation-induced DNA damage was sequence specific in both short DNA sequences and the entire human genome. The degree of damage at each nucleotide was quantified and it was found that the precise location of the damage site was influenced by the surrounding sequence context. This resulted in a consensus sequence of WWNGG*G in short DNA sequences and GSC*M (where M is A or C) in genomic DNA. A comparative analysis revealed an overall consensus sequence of GGSC*MC. The consistency of the consensus sequence demonstrated that damage sites are specific and non- random in nature