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Recently, hydrogen peroxide (H₂O₂), and its free-radical product the hydroxyl radical (OH·), have been identified as major sources of DNA damage in living organisms. We examined DNA repair of hydrogen peroxide damage, using a standard bacteriophage T4 test system in which several different types of repair could be determined. Post-replication recombinational repair and denV-dependent excision repair had little or no effect on H₂O₂ damage. Also, an enzyme important in repair of H₂O₂-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H₂O₂ damage to the phage. However, multiplicity reactivation, a form of recombinational repair between multiply infecting phage genomes, was found to repair H₂O₂ damages efficiently. The RAD52 gene of Saccharomyces cerevisiae and genes 46 and 47 of bacteriophage T4 are essential for most recombination and recombinational repair in their respective organisms. The RAD52 gene was introduced into expression vectors which were used to transform E. coli. RAD52 expression was induced, and its ability to complement either gene 46 or gene 47 phage mutants was determined with respect to phage growth, recombination, and recombinational repair. RAD52 gene expression allowed growth of gene 46 and gene 47 mutants under otherwise restrictive conditions, as measured by plaque formation and burst size. The RAD52 gene also restored the ability of gene 46 and gene 47 mutants to undergo recombination of rII markers. Furthermore, the RAD52 gene restored recombinational repair after UV irradiation of gene 46 and gene 47 mutants. The published DNA sequence of RAD52 was compared with the published sequences of genes 46 and 47. Although overall homologies were only marginally significant, RAD52 and gene 46 had substantial sequence similarity over a limited region. These results indicate that the recombinational repair pathway found in phage T4 may be ubiquitous for DNA damage caused by endogenous exidative reactions. Furthermore, they indicated that an essential element of the recombination mechanism in both procaryotic viruses and eucaryotes arose from a common ancestor. Procaryotes and eucaryotes are thought to have diverged at least one billion years ago. Thus, recombination apparently arose early in evolution.

The DNA sequence in every human being is a text of three billion characters from a four letter alphabet; determining this sequence is a major project in molecular biology. The fundamental task biologists face is to reconstruct a long sequence given short fragments from unknown locations. These fragments contain errors, and may represent the sequence on one strand of the double-helix, or the reverse complement sequence on the other strand. The Sequence Reconstruction Problem is, given a collection F of fragment sequences and an error rate 0 ≤ ε < 1, find a shortest sequence S such that every fragment F ∈ F, or its reverse complement, matches a substring of S with at most ε|F| errors. Sequence Reconstruction is NP-complete. We decompose the problem into (1) constructing a graph of approximate overlaps between pairs of fragments, (2) selecting a set of overlaps of maximum total weight that induce a consistent layout of the fragments, (3) merging the overlaps into a multiple sequence alignment and voting on a consensus. A solution to (1) through (3) yields a reconstructed sequence feasible at error rate 2ε/(1-ε) and at most a factor 1/1-ε longer than the shortest reconstruction, given some assumptions on fragment error. We define a measure of the overlap in a reconstruction, show that maximizing the overlap minimizes the length, and that approximating (2) within a factor of α approximates Sequence Reconstruction within a factor of (1- ε)α under the overlap measure. We construct the overlap graph for (1) in O(εN²) time given fragments of total length N at error rate ε. We develop two exact and two approximation algorithms for (2). Our best exact algorithm computes an optimal layout for a graph of E overlaps and V fragments in O(K(E + V log V)) time, where K ≤ 2ᴱ is the size of the branch-and-bound search tree. Our best approximation algorithm computes a layout with overlap at least 1/2 the maximum in O(V(E + V log V)log V) time. This is the first treatment of Sequence Reconstruction with inexact data and unknown complementarity.

Members of multi-specific DNA-binding protein families have evolved to specifically recognize diverse DNA site sequences. This dissertation presents evidence that the Cro protein family of helix-turn-helix transcription factors from lambdoid bacteriophages may share a conserved, limited "code" that partially governs evolution of their binding specificity. A bioinformatic study revealed six conserved sequence correlations between residues at three positions in Cro recognition helices and three base-pairs in putative cognate DNA consensus half-sites (Chapter 2). Three of these pairings correspond to sequence-specific contacts observed at the binding interface of lambda Cro and consensus operator DNA in a previously available co-crystal structure (Albright and Matthews, 1998a). In vitro mutagenesis and functional characterization was used to validate the proposed "code" (Chapter 3). Two out of three "coding" combinations acted as specificity switches in lambda Cro, though variant proteins displayed reduced binding specificity for their predicted target DNA sites. Two crystal structures of a lambda Cro variant are presented in Chapter 4, which provide insight into lambda Cro dimer flexibility. Additionally, a co-crystal structure of N15 Cro bound to consensus site DNA was determined which contains two coding residue pairs at the binding interface (Chapter 5), and a crystal structure of Xfasa1 Cro that enables future investigations into Cro functional evolution (Chapter 6). Although there are several caveats, the data are consistent with a model in which Cro proteins may indeed have evolved new binding specificities in part through simple mutations at their binding interfaces that follow a simple set of evolutionarily conserved "coding" rules. The structural and functional diversity of Cro proteins provides an exciting venue for future research into their evolution.

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