A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER.

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a 'closed' inactive state that needs to undergo a major rearrangement to adopt an 'open' active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans.

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5' and 3' sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

The three Endonuclease III variants of Deinococcus radiodurans possess distinct and complementary DNA repair activities.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase that removes oxidized pyrimidines from DNA. The genome of Deinococcus radiodurans encodes for an unusually high number of DNA glycosylases, including three EndoIII enzymes (drEndoIII1-3). Here, we compare the properties of these enzymes to those of their well-studied homologues from E. coli and human. Our biochemical and mutational data, reinforced by MD simulations of EndoIII-DNA complexes, reveal that drEndoIII2 exhibits a broad substrate specificity and a catalytic efficiency surpassing that of its counterparts. In contrast, drEndoIII1 has much weaker and uncoupled DNA glycosylase and AP-lyase activities, a characteristic feature of eukaryotic DNA glycosylases, and was found to present a relatively robust activity on single-stranded DNA substrates. To our knowledge, this is the first report of such an activity for an EndoIII. In the case of drEndoIII3, no catalytic activity could be detected, but its ability to specifically recognize lesion-containing DNA using a largely rearranged substrate binding pocket suggests that it may play an alternative role in genome maintenance. Overall, these findings reveal that D. radiodurans possesses a unique set of DNA repair enzymes, including three non-redundant EndoIII variants with distinct properties and complementary activities, which together contribute to genome maintenance in this bacterium.