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DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn2 + but not Mg2 + significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn2 + provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.

The single-crystal structures are presented for two DNA sequences with the thymine bases covalently cross-linked across the complementary strands by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT). The HMT-adduct of d(CCGCTAGCGG) forms a psoralen-induced Holliday junction, showing for the first time the effect of this important class of chemotheraputics on the structure of the recombination intermediate. In contrast, HMT-d(CCGGTACCGG) forms a sequence-dependent junction. In both structures, the DNA duplex is highly distorted at the thymine base linked to the six-member pyrone ring of the drug. The psoralen cross-link defines the intramolecular interactions of the drug-induced junction, while the sequence-dependent structure is nearly identical to the native Holliday junction of d(CCGGTACCGG) alone. The two structures contrast the effects of drug- and sequence-dependent interactions on the structure of a Holliday junction, suggesting a role for psoralen in the mechanism to initiate repair of psoralen-lesions in mammalian DNA.

We have solved the structures of the homoduplex d(Gm5CGCGCG)2, and the heteroduplexes d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/d(CCGCGCG). The structures form six base-pairs of identical Z-DNA duplexes with single nucleotides overhanging at the 5'-ends. The overhanging nucleotide from one strand remains stacked and sandwiched between the blunt-ends of two adjacent Z-DNA duplexes, while the overhanging base of the opposing strand is extra-helical. The stacked and the extra-helical bases from adjacent duplexes pair to form a distorted d(G x G) reverse Hoogsteen base-pair in the d(Gm5CGCGCG)2 homoduplex, and d(G x T) reverse wobble and d(G x C) reverse Watson-Crick base-pairs in the d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/d(CCGCGCG) heteroduplexes, respectively. Interestingly, only the d(G,T) and d(G x C) base-pairs were observed in the heteroduplexes, suggesting that both the d(G x T) reverse wobble and d(G x C) reverse Watson-Crick base-pairs are more stable in this crystal environment than the d(G x G) reverse Hoogsteen base-pair. To estimate the relative stability of the three types of reverse base-pairs, crystals were grown using various mixtures of sequences and their strand compositions analyzed by mass spectrometry. The d(G x C) reverse Watson-Crick base-pair was estimated to be more stable by approximately 1.5 kcal/mol and the d(G x T) reverse wobble base-pair more stable by approximately 0.5 kcal/mol than the d(G x G) reverse Hoogsteen base-pair. The step during crystallization responsible for discriminating between the strands in the crystal is highly cooperative, suggesting that it occurs during the initial nucleating event of crystal growth.

DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from from those of other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.