Abstract
Homologous recombination is a widely conserved process by which identical sequences of DNA cross over. This process is essential during cell division and increases genetic diversity. Homologous recombination is also an important mechanism in repairing DNA damage. In order for recombination to occur the cell must find a sequence of DNA identical to the one it must replace. Homologous recombination is carried out by and affected by a number of genes. RecA is the main factor in involved in homologous recombination as well as the DNA repair system. RecA loading onto the DNA is mediated by two different pathways, RecBCD and RecFOR. RecBCD recognizes double stranded break sand RecFOR pathway preferentially identifies single stranded gaps in DNA. Along with RecA, RecN plays a vital role in homologous recombination as it tethers DNA strands together during recombination. Two other vital genes involved in homologous recombination are RecG and RuvC, both which play important roles in resolving the Holliday junctions formed during recombination. There are also various mutagenic drugs that have an effect on DNA repair and homologous recombination mechanisms, such as AZT, 5- Azacytidine, Formaldehyde, and Ciprofloxacin. Using a lacZ reporter system and blue white screening we have developed a way in which to study homologous recombination at varying distances. Two chromosomal constructs have been created; one where a recipient LacZ has had a 5bp active site gene sequence deleted, this deletion creates an inactive LacZ protein and a donor sequence containing this 5bp deletion has been inserted 24minutes apart, at the attTn7 with 250bp of homology on either side of this 5bp active site. The second construct involves the complete deletion of the LacZ gene from its natural locus, and both the recipient, LacZ with the 5bp active site deletion, and donor sequences, the 5bp active site with 250bp homology on either side, have been inserted at the attTn7 site on the chromosome. These two constructs allow for the study of distance on homologous recombination both in regard to the genes involved in recombination that have been deleted, as well as to the portion of these constructs that were exposed to the various mutagenic drugs.