Holliday junctions (HJs) are central intermediates involved in a variety of DNA repair and recombination pathways. In phage lambda Integrase (Int) protein-mediated site-specific recombination (SSR), the isomerization of Int-bound HJ determines the directionality of HJ resolution reaction and results in the entry of phage lysogentic cycle or lytic cycle. Previously, it was found that this directional bias could be altered by 3 mutated nucleotide pairs at the center of HJ. Three consecutive C-G pairs (BS substrate) lead the HJ resolution to recombination products, while 3 A-T pairs (TS substrate) lead to recombination substrates. In the first chapter of this work, we first examined the effect of the sequence mutations on excisive recombination and found both mutated substrates showed significant decrease in recombination efficiency. To further determine how different mutations affect the kinetics of the excisive recombination, we then combined biochemical assay (e.g. HJ resolution) with mathematical modeling approaches to identify the kinetic constants of each of the reaction steps. Result shows that the first round of catalysis event is not only the fastest step during the wild-type (WT) HJ resolution but is also the most affected step when sequence mutations occur in the center of HJs. Moreover, since BS substrate shows 6X increase on the rate of the first catalysis event while rates of the rest of the steps in the resolution reaction remain similar to WT, it suggests that the poor efficiency of BS excisive recombination can only be the result of a deficient presynapsis and/or synapsis step. In the second chapter, several HJ-trapping peptides and small molecules identified previously in our lab were tested for their ability to inhibit the resolution of HJ mediated by the resolvase of fowlpox virus (FPV), a member in the family Poxviridae. Poxviruses are notorious for causing a variety of skin and mucosal infectious diseases and one of its members, variola virus, has caused a new concern as it may be used as a weapon for bioterrorism. Poxvirus resolvases, as one of the essential enzymes of Poxviruses, are members of the RNase H superfamily and they resolve the concatemeric chromosome to unit-length chromosome during the viral replication. Here we present the most potent peptide identified, KWWCRW, which inhibits in vitro DNA cleavage mediated by FPV resolvase and competes with FPV resolvase for the binding site on HJ. Peptide WRWYCR and small molecules TPI 1609-10, 1609-12, 1795-10, and 1795-12 were also identified as potent inhibitors with lesser potency than KWWCRW.