Kendall L. Knight

H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form γ-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that γ-H2AX spreads a limited distance up to 1–2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of γ-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of γ-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.

Molecular Design and Functional Organization of the RecA Protein

ABSTRACT The bacterial RecA protein participates in a remarkably diverse set of functions, all of which are involved in the maintenance of genomic integrity. RecA is a central component in both the catalysis of recombinational DNA repair and the regulation of the cellular SOS response. Despite the mechanistic differences of its functions, all require formation of an active RecA/ATP/DNA complex. RecA is a classic allosterically regulated enzyme, and ATP binding results in a dramatic increase in DNA binding affinity and a cooperative assembly of RecA subunits to form an ordered, helical nucleoprotein filament. The molecular events that underlie this ATP-induced structural transition are becoming increasingly clear. This review focuses on descriptions of our current understanding of the molecular design and allosteric regulation of RecA. We present a comprehensive list of all published recA mutants and use the results of various genetic and biochemical studies, together with available structural information, to develop ideas regarding the design of RecA functional domains and their catalytic organization.

Discovery of a Novel Function for Human Rad51: MAINTENANCE OF THE MITOCHONDRIAL GENOME*

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.

Cellular Redistribution of Rad51 in Response to DNA Damage NOVEL ROLE FOR Rad51C

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51

Rad51‐mediated homologous recombination (HR) is essential for maintenance of genome integrity. The Xrcc3 protein functions in HR DNA repair, and studies suggest it has multiple roles at different stages in this pathway. Defects in vertebrate XRCC3 result in elevated levels of spontaneous and DNA damage‐induced chromosomal abnormalities, as well as increased sensitivity to DNA damaging agents. Formation of DNA damaged‐induced nuclear Rad51 foci requires Xrcc3 and the other Rad51 paralog proteins (Rad51B, Rad51C, Rad51D, Xrcc2), thus supporting a model in which an early function of Xrcc3 involves promoting assembly of active Rad51 repair complexes. However, it is not known whether Xrcc3 or other Rad51 paralog proteins accumulate at DNA breaks, and if they do whether their stable association with breaks requires Rad51. Here we report for the first time that Xrcc3 forms distinct foci in human cells and that nuclear Xrcc3 begins to localize at sites of DNA damage within 10 min after radiation treatment. RNAi‐mediated knock down of Rad51 has no effect on the DNA damage‐induced localization of Xrcc3 to DNA breaks. Our data are consistent with a model in which Xrcc3 associates directly with DNA breaks independent of Rad51, and subsequently facilitates formation of the Rad51 nucleoprotein filament.

Allosteric Regulation of RecA Protein Function Is Mediated by Gln194

Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical “allosteric switch” for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.

Phe217 Regulates the Transfer of Allosteric Information across the Subunit Interface of the RecA Protein Filament

BACKGROUND ATP-mediated cooperative assembly of a RecA nucleoprotein filament activates the protein for catalysis of DNA strand exchange. RecA is a classic allosterically regulated enzyme in that ATP binding results in a dramatic increase in ssDNA binding affinity. This increase in ssDNA binding affinity results almost exclusively from an ATP-mediated increase in cooperative filament assembly rather than an increase in the inherent affinity of monomeric RecA for DNA. Therefore, certain residues at the subunit interface must play an important role in transmitting allosteric information across the filament structure of RecA. RESULTS Using electron microscopic analysis of RecA polymer formation in the absence of DNA, we show that while wild-type RecA undergoes a slight decrease in filament length in the presence of ATP, a Phe217Tyr substitution results in a dramatic ATP-induced increase in cooperative filament assembly. Biosensor DNA binding measurements reveal that the Phe217Tyr mutation increases ATP-mediated cooperative interaction between RecA subunits by more than 250-fold. CONCLUSIONS These studies represent the first identification of a subunit interface residue in RecA (Phe217) that plays a critical role in regulating the flow of ATP-mediated information throughout the protein filament structure. We propose a model by which conformational changes that occur upon ATP binding are propagated through the structure of a RecA monomer, resulting in the insertion of the Phe217 side chain into a pocket in the neighboring subunit. This event serves as a key step in intersubunit communication leading to ATP-mediated cooperative filament assembly and high affinity binding to ssDNA.

Cellular localization of human Rad51C and regulation of ubiquitin-mediated proteolysis of Rad51

Rad51‐catalyzed homologous recombination is an important pathway for repair of DNA double strand breaks and maintenance of genome integrity in vertebrate cells. Five proteins referred to as Rad51 paralogs promote Rad51 activity and are proposed to act at various, and in some cases, multiple stages in the recombination pathway. Imaging studies of native Rad51 have revealed its cellular response to DNA damage, yet visualization of the paralog proteins has met with limited success. In this study, we are able to detect endogenous Rad51C and Xrcc3 in human cells. In an effort to determine how Rad51, Rad51C, and Xrcc3 influence the pattern of localization of each other over the time course of DNA damage and repair, we have made the unexpected observation that Rad51 degradation via the ubiquitin‐mediated proteasome pathway occurs as a natural part of recombinational DNA repair. Additionally, we find that Rad51C plays an important role in regulating this process. This article contains supplementary material, which may be viewed at the Journal of Cellular Biochemistry website at http://www.interscience.wiley.com/jpages/0730‐2312/suppmat/index.html.

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly‐157 and Glu‐158 are ‘hot‐spots’ for the occurrence of constitutive LexA co‐protease mutants (coprtc). In the present study, we clearly establish that position 157 is a hot‐spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co‐protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu‐154→Asp and Glu‐154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second‐site mutations that completely suppress coprtc activity toward LexA have little or no effect on the coprtc activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second‐site suppressor mutations, suggesting a functional interaction among side‐chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co‐protease substrate‐binding site.

Human Rad51 promotes mitochondrial DNA synthesis under conditions of increased replication stress.

Homologous recombination is essential for productive DNA replication particularly under stress conditions. We previously demonstrated a stress-induced recruitment of Rad51 to mitochondria and a critical need for its activity in the maintenance of mitochondrial DNA (mtDNA) copy number. Using the human osteosarcoma cell line U20S, we show in the present study that recruitment of Rad51 to mitochondria under stress conditions requires ongoing mtDNA replication. Additionally, Rad51 levels in mitochondria increase in cells recovering from mtDNA depletion. Our findings highlight an important new role for Rad51 in supporting mtDNA replication, and further promote the idea that recombination is indispensable for sustaining DNA synthesis under conditions of replication stress.