A Partially Functional DNA Helicase II Mutant Defective in Forming Stable Binary Complexes with ATP and DNA: A ROLE FOR HELICASE MOTIF III

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed

A Point Mutation in Escherichia coli DNA Helicase II Renders the Enzyme Nonfunctional in Two DNA Repair Pathways: EVIDENCE FOR INITIATION OF UNWINDING FROM A NICKIN VIVO

Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations. These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins. Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. To define further the role of UvrD in DNA repair a site-specific mutant was characterized. The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases. The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair. Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick. The elongation phase of the unwinding reaction was nearly normal. Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis. These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair

Inhibition of Werner Syndrome Helicase Activity by Benzo[ c ]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent

Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (-)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3' or the 5' side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts

FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro , and Its Catalytic Activities Are Regulated by Phosphorylation

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb

DNA Replication Stress

This Special Issue of International Journal of Molecular Sciences (IJMS) is dedicated to the mechanisms mediated at the molecular and cellular levels in response to adverse genomic perturbations and DNA replication stress. The relevant proteins and processes play paramount roles in nucleic acid transactions to maintain genomic stability and cellular homeostasis. A total of 18 articles are presented which encompass a broad range of highly relevant topics in genome biology. These include replication fork dynamics, DNA repair processes, DNA damage signaling and cell cycle control, cancer biology, epigenetics, cellular senescence, neurodegeneration, and aging. As Guest Editor for this IJMS Special Issue, I am very pleased to offer this collection of riveting articles centered on the theme of DNA replication stress. The blend of articles builds upon a theme that DNA damage has profound consequences for genomic stability and cellular homeostasis that affect tissue function, disease, cancer, and aging at multiple levels and through unique mechanisms. I thank the authors for their excellent contributions, which provide new insight into this fascinating and highly relevant area of genome biology