Ca2+/calmodulin-dependent protein kinase-II (CaMKII) is unique among protein kinases for its dodecameric assembly and its complex response to Ca2+. The crystal structure of the autoinhibited kinase domain of CaMKII, determined at 1.8 A resolution, reveals an unexpected dimeric organization in which the calmodulin-responsive regulatory segments form a coiled-coil strut that blocks peptide and ATP binding to the otherwise intrinsically active kinase domains. A threonine residue in the regulatory segment, which when phosphorylated renders CaMKII calmodulin independent, is held apart from the catalytic sites by the organization of the dimer. This ensures a strict Ca2+ dependence for initial activation. The structure of the kinase dimer, when combined with small-angle X-ray scattering data for the holoenzyme, suggests that inactive CaMKII forms tightly packed autoinhibited assemblies that convert upon activation into clusters of loosely tethered and independent kinase domains.

Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.disulfide cross-linking | DNA translocation | protein-DNA structure | molecular dynamics simulations D amage to the covalent structure of DNA arising from spontaneous degradation, replication errors, and the attack of exogenous and endogenous genotoxins undermines the genomic integrity and hence the viability of all life forms (1). As the first line of defense against many of these genome insults, DNA glycosylases recognize aberrant nucleobases in DNA and catalyze scission of their glycosidic bond, thereby initiating correction of the damage through the base excision DNA repair pathway (2). Failure to repair DNA lesions has deleterious consequences to the organism (3), and yet searching for the lesions represents one of the most formidable needle-in-the-haystack problems in biology (4). First, many damaged nucleobases resemble closely their millionfold more abundant undamaged progenitors, leaving little in the way of a damage signature for recognition. Secondly, all known DNA glyclosylases must extrude the target lesion from DNA and insert it into an extrahelical active site pocket to perform glycosidic bond cleavage, which requires significant energy input with lesions that do not destabilize the DNA duplex. Lastly, DNA glycosylases utilize only thermal energy to drive a directionally unbiased, massively redundant Brownian search for target lesions. High-resolution structures of various DNA glycosylases in complex with lesion-containing or normal DNA duplexes (5-12) have provided important insights into the means by which these enzymes recognize lesions, promote extrusion of lesions from DNA, and catalyze the chemical steps of base excision. However, virtually nothing is known about the atomic details of how DNA glycosyalses achieve nearly barrierless translocation along DNA (13).The DNA repair protein for which the processes of lesion search and damage recognition are most well-understood at the atomic (5-7, 12, 14-17) and biochemical (18-20) levels is MutM (also known as Fpg), a bact...

Background:The role of helix-invading residues during MutM-catalyzed oxoG repair remains unclear.

Background:The molecular basis for sequence-dependent variation in DNA repair is poorly understood. Results: A systematic study of lesion encounter by MutM reveals major differences in stacking of the target oxoG. Conclusion: Sequence-dependent changes in base stacking may contribute to lesion extrusion and repair. Significance: This is the first structural study of sequence context effects on lesion recognition for a DNA repair enzyme.

Nucleotide excision repair (NER) is an essential DNA repair system distinguished from other such systems by its extraordinary versatility. NER removes a wide variety of structurally dissimilar lesions having only their bulkiness in common. NER can also repair several less bulky nucleobase lesions, such as 8-oxoguanine. Thus, how a single DNA repair system distinguishes such a diverse array of structurally divergent lesions from undamaged DNA has been one of the great unsolved mysteries in the field of genome maintenance. Here we employ a synthetic crystallography approach to obtain crystal structures of the pivotal NER enzyme UvrB in complex with duplex DNA, trapped at the stage of lesion-recognition. These structures coupled with biochemical studies suggest that UvrB integrates the ATPase-dependent helicase/translocase and lesion-recognition activities. Our work also conclusively establishes the identity of the lesion-containing strand and provides a compelling insight to how UvrB recognizes a diverse array of DNA lesions.

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