Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals

Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling

Mechanistic insights into Xer recombination and conjugative transposition in Helicobacter pylori

Site-specific recombinases of the Xer family are essential in most bacteria with circular chromosomes for the resolution of chromosome dimers arising after genome replication. In Helicobacter pylori, a gastric pathogen implicated in peptic ulcer disease and gastric cancer, the chromosome dimers are resolved by a single Xer recombinase, XerH. Interestingly, many H. pylori strains carry a second Xer recombinase, XerT, usually encoded on a large conjugative transposon TnPZ. Remarkably, XerT is not involved in chromosome dimer resolution, but was shown to be required for the mobilization of TnPZ in vivo. In this thesis, I investigated the molecular mechanisms of XerH- and XerT-mediated recombination by combining X-ray crystallography with protein biochemistry and microbiology. I solved the crystal structure of the XerH tetramer in a post-cleavage synaptic complex with its substrate DNA site, difH. To our knowledge, this is the first structure of an Xer recombinase bound to DNA. The structure and additional biochemical data provided key insights into the ordering and regulation of difH binding and first strand cleavage by XerH. Moreover, I investigated the regulation of XerH recombination by FtsK – a host factor usually required for Xer recombination – and found that XerH can resolve plasmids in the absence of FtsK in E. coli, but additional factors might be required for recombination of chromosome-borne difH sites. In the second part of this work, I studied the mechanism of XerT-mediated TnPZ transposition. XerT’s binding and cleavage sites on transposon ends were mapped and XerT activity was reconstituted in vitro by trapping cleavage and strand exchange products. In addition, the complete TnPZ excision has been reconstituted in vivo in E. coli, indicating that XerT is sufficient to catalyze TnPZ mobilization. Based on the results, a testable model for TnPZ excision and integration was proposed. In summary, this work provides valuable insights into the mechanisms of the two Xer recombinases of H. pylori and enhances our understanding of Xer recombination (a process essential for bacterial survival) and conjugative transposition (important in the spread of antibiotic resistance among bacteria), which in the future could help develop new therapeutic agents against deadly pathogens such as H. pylori or help control the spread of antibiotic resistance

A novel DNA primase-helicase pair encoded by SCCmec elements

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3'-to-5' helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec

A novel dna primase-helicase pair encoded by SCC<i>mec</i> elements

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here we biochemically characterize 3 proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3’-to-5’ helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol MP. Taken together, our results suggest that this is a functional replication module for SCCmec

Targeting IS<i>608</i> transposon integration to highly specific sequences by structure-based transposon engineering

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS 608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction net- work between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.</p