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Abstract
The antitumor activity of bleomycin is thought to arise from its ability to mediate DNA damage. Previous studies established GT and GC sequences as the major sites of bleomycin cleavage. Adjacent nucleotides are also known to subtly influence bleomycin preference for a particular site—however, this has yet to be characterised in detail. Furthermore, analogues of bleomycin often cleave at similar sites, but their relative preference can differ.
This thesis aims to examine the extended sequence specificity of bleomycin—and compare it to related analogues. Understanding the subtle differences in the sequence specificity of the bleomycins could help elucidate their mechanism of action—and subsequently the rational design of more effective analogues.
We utilised two different approaches—first, the RTGTAY plasmid construct was designed to contain 32 motifs with the sequence XXGTAX, where the X nucleotides were systematically varied to allow for subtle quantifications. Second, we utilised next-generation sequencing methods to detect bleomycin cleavage sites in the entire human genome—and in cellular environments. Previous studies often utilised short purified DNA sequences that did not permit the importance of complex cellular components to be evaluated. Using next-generation sequencing allowed us to examine bleomycin cleavage over a vast combination of DNA sequences—generating over 200 million cleavage sites per sample, enabling detailed characterisation of the bleomycin sequence specificity in human cellular and purified DNA environments.
Utilising Illumina next-generation sequencing, we were able to rank DNA motifs based on their frequency of cleavage by bleomycin. We show that the strongest cleaved sites contained the core sequence TGTA. Interestingly, there were slight differences in the bleomycin specificity on purified genomic DNA, compared to cellular. Additionally, the results from the RTGTAY construct closely mirror that of the purified genomic DNA.
We also utilised the RTGTAY plasmid construct to compare the extended sequence specificity of two novel analogues—6′-deoxy-BLM Z and BLM Z, compared with bleomycin and zorbamycin. These novel analogues have a similar sequence specificity compared with bleomycin. However, zorbamycin exhibited a very different cleavage profile. The characterisation of bleomycin extended sequence specificity could help evaluate the functional properties of its related analogues.