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    Kendall L Knight PhD

    TitleProfessor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentBiochemistry and Molecular Pharmacology
    AddressUniversity of Massachusetts Medical School
    55 Lake Avenue North
    Worcester MA 01655
    Phone508-856-2405
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentBiochemistry and Molecular Pharmacology

      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentInterdisciplinary Graduate Program

        Overview 
        Narrative

        Academic Background

        Dr. Knight received his Ph.D. from the Department of Biochemistry at U.C. Riverside in 1981. He worked as an American Cancer Society post-doctoral fellow with Kevin McEntee at the U.C.L.A. School of Medicine from 1981-1985, and was awarded a Charles A. King Trust research fellowship for post-doctoral work with Bob Sauer in the Department of Biology at M.I.T. from 1985-1989.

         

        Associate Dean of the GSBS Basic and Biomedical Sciences Division & UMMS Assistant Vice-Provost of Admissions

         

        Homologous recombinational DNA repair in human cells

         

        Photo: Kendall KnightWe are interested in understanding the catalysis and regulation of Rad51-mediated DNA repair in human cells.  Our research involves biochemical and cell biological studies of Rad51, as well as the contribution of various Rad51-interacting proteins to the overall mechanism of Rad51 function, e.g. Rad51C, Xrcc3, Brca2, etc.  We are also engaged in biochemical and biophysical studies of the molecular design of proteins that participate in the catalysis of homologous genetic recombination and recombinational DNA repair.  Our research takes advantage of a number of experimental approaches in genetics,RNAi, cell biology, microscopy, molecular biology, protein chemistry/enzymology and biophysics aimed at providing a molecular and cellular description of the catalytic organization of the Rad51 protein and Rad51 protein complexes.  

        Human Rad51 and related proteins

        The majority of our work focused on studies of the coordinated action of the human Rad51 and the Rad51 paralog proteins (Xrcc3 and Rad51C).  In our initial work we took advantage of several improvements we made in immunofluorescence methods to show that Rad51, Rad51C, and Xrcc3 have a significant cytoplasmic presence in human cells, and that as a function of DNA damage Rad51 and Rad51C are re-distributed toward the nuclear periphery and into the nucleus (Figs. 1 & 2, and see Forget, Bennett & Knight, 2004; Bennett & Knight, 2005).  Further studies revealed that Rad51C contributes to a DNA damage-induced and BRCA2-independent increase in nuclear levels of Rad51 (Gildemeister, Sage & Knight, 2009).  An unexpected outcome of these studies was our discovery that human Rad51, Rad51C and Xrcc3 function in mitochondria to maintain mtDNA copy number in response to DNA damage.  We found that DNA damage results in increased mitochondrial levels of each protein as well as an increased physical association of Rad51 with mtDNA (Sage, Gildemeister & Knight, 2010).  Additionally, we showed that Rad51 promotes mtDNA synthesis under conditions of replication stress in either the presence or absence of DNA damage (Sage & Knight, 2013).

        Rad51 cellular localization as a function of DNA damage Rad51C cellular localization during a time course of DNA damage and repair
             Figure 1     Figure 2

         

        ATP binding by the Rad51 K133A proteinFor our mutagenesis studies of human Rad51 we are creating single and multiple mutations in targeted domains, e.g. the ATP and DNA binding sites, the oligomeric interface, protein-protein interaction domains, etc. to identify important determinants of different aspects of protein structure and function.  Specific mutant proteins are chosen to analyze using cell-based recombination and DNA repair assays, and for purification and detailed in vitro analyses of their biochemical and structural properties.  Our recent study of mutations in the Rad51 ATP binding site revealed an unexpected result that, in fact, supports a long-standing model for how recombinase enzymes such as Rad51 and bacterial RecA use the energy derived from ATP binding and turnover/release to promote recombination.  We found that a mutant Rad51 carrying a Lys-to-Ala substitution in the Walker A motif of the ATP binding site was partially functional in a cell-based DNA repair assay (see Forget et al., 2007).  In this work we showed that the purified protein binds ATP but with an affinity approximately100-fold lower than wild type Rad51 (Fig. 3).  This was important because it is commonly assumed that this mutation prohibits ATP binding in any ATP-binding protein into which it is introduced.  Therefore, our data supports a model in which ATP binding promotes nucleoprotein filament assembly and function in the recombination process, while ATP hydrolysis and release of ADP (or simple release of non-hydrolyzed ATP in the case of the Lys-to-Ala mutant protein) promotes filament disassembly.

        4Pi microscopy

        We published the first successful use of high-resolution 4Pi microscopy to visualize endogenous nuclear proteins (see Bewersdorf, Bennett & Knight, 2006).  4Pi microscope technology was created in the laboratory of Stefan W. Hell, and offers a significant improvement in z-axis resolution (the optical axis).  The illuminating light from two perfectly aligned opposing objective lenses interferes constructively within the sample, thereby improving resolution (Fig. 4).  A similar constructive interference of the collected fluorescent light adds to the increase in resolution, providing an approximate 6-fold improvement in z-axis resolution over standard confocal microscopes.  http://www.mpibpc.gwdg.de/groups/hell/4Pi.htm

        Histone H2AX imaging using 4Pi vs. confocal microscopy

        Our first efforts were directed at imaging histones H2AX and g-H2AX, a phosphorylated version of H2AX that appears immediately following DNA damage and serves as a mark for the site of a DNA break.  This work supports a model in which chromatin structures carrying clusters of H2AX serve as platforms for the immediate response to DNA damage, and also serve to limit the spread of H2AX phosphorylation (Figs. 5 & 6; movies 1 & 2; see Bewersdorf, Bennett & Knight 2006).

        4Pi microscope images of H2AX and gH2AX following DNA damage

        Cluster size of H2AX and gH2AX chromatin structures as a function of time after DNA damage

        Movie One
        Three-dimensional video representation of a 4Pi microscopy data set showing nuclear distribution of H2AX and g-H2AX at 15 min after exposure of cells to ionizing radiation. The movie is based on the same data set used in Fig. 1 (15 min) and was created with the rendering software Amira. H2AX is in green and g -H2AX is in red. The grid has a 1-m m raster and axes are as indicated in Fig. 1 in the full manuscript (Bewersdorf et al. 2006).

        Movie Two
        Three-dimensional video representation of a 4Pi microscopy data set showing nuclear distribution of H2AX and g-H2AX at 180 min after exposure of cells to ionizing radiation. The movie is based on the same data set used in Fig. 1 and was created with the rendering software Amira. H2AX is in green and g -H2AX is in red. The grid has a 1-m m raster and axes are as indicated in Fig. 1 in the full manuscript (Bewersdorf et al. 2006).

        Bacterial RecA

        Our studies of genome stability in human cells and Rad51-mediated DNA repair derive from work on the related bacterial RecA protein.  Both RecA and human Rad51 bind to single-stranded regions at DNA breaks and form a helical nucleoprotein filament, which then initiates strand exchange and transfer of information between homologous DNA molecules, most frequently a sister chromatid following DNA replication (McGrew & Knight, 2003).  RecA and human Rad51 have similar structural and functional properties, but also show distinct differences in their structure-function relationships (see De Zutter & Knight, 1999).  We showed that Phe217 is a critical subunit interface residue in RecA that regulates the flow of ATP-mediated allosteric information throughout the protein filament (see De Zutter et al., 2001).  However, the human Rad51 protein does not have an aromatic residue conserved at this position, and any ATP-mediated allosteric effect on its overall function appears to be significantly more subtle than that observed for RecA (Fig 3 above; see Forget et al., 2007).

        In our final project with bacterial RecA, we addressed a long-standing question in the recombination field – what is the protein unit that initiates filament assembly on ssDNA?  Because purified RecA protein exists as a heterogeneous mix of various oligomeric states, e.g. monomer, dimer, trimer, hexamer, short filaments, etc., it has been impossible to confirm the original idea that a monomer of RecA is the nucleating unit for filament assembly.  However, we have recently published a study in which we created a fused RecA dimer and showed that this protein is perfectly capable of forming filaments on ssDNA and catalyzing recombinational DNA repair (see Forget et al., 2006).  Therefore, in the cell, where it is important for RecA to respond immediately to DNA damage, it is very likely that a mixture of monomers, as well as pre-formed dimers and perhaps trimers of RecA nucleate filament formation.

        In addition to providing a detailed molecular understanding of homologous genetic recombination, and the mechanisms by which multifunctional, oligomeric enzymes and protein complexes coordinate a variety of activities to carry out the ordered enzymatic process, our work will offer insights into processes critical for maintenance of genome stability. 

        Movie One

        Movie Two


         



        Bibliographic 
        selected publications
        List All   |   Timeline
        1. Sage JM, Knight KL. Human Rad51 promotes mitochondrial DNA synthesis under conditions of increased replication stress. Mitochondrion. 2013 Jul; 13(4):350-6.
          View in: PubMed
        2. O'Brien SK, Knight KL, Rana TM. Phosphorylation of histone H1 by P-TEFb is a necessary step in skeletal muscle differentiation. J Cell Physiol. 2012 Jan; 227(1):383-9.
          View in: PubMed
        3. Bakhlanova IV, Dudkina AV, Baitin DM, Knight KL, Cox MM, Lanzov VA. Modulating cellular recombination potential through alterations in RecA structure and regulation. Mol Microbiol. 2010 Dec; 78(6):1523-38.
          View in: PubMed
        4. Sage JM, Gildemeister OS, Knight KL. Discovery of a novel function for human Rad51: maintenance of the mitochondrial genome. J Biol Chem. 2010 Jun 18; 285(25):18984-90.
          View in: PubMed
        5. Gildemeister OS, Sage JM, Knight KL. Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C. J Biol Chem. 2009 Nov 13; 284(46):31945-52.
          View in: PubMed
        6. Bennett BT, Bewersdorf J, Knight KL. Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy. Methods. 2009 May; 48(1):63-71.
          View in: PubMed
        7. Forget AL, Loftus MS, McGrew DA, Bennett BT, Knight KL. The human Rad51 K133A mutant is functional for DNA double-strand break repair in human cells. Biochemistry. 2007 Mar 20; 46(11):3566-75.
          View in: PubMed
        8. Bewersdorf J, Bennett BT, Knight KL. H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy. Proc Natl Acad Sci U S A. 2006 Nov 28; 103(48):18137-42.
          View in: PubMed
        9. Forget AL, Kudron MM, McGrew DA, Calmann MA, Schiffer CA, Knight KL. RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments. Biochemistry. 2006 Nov 14; 45(45):13537-42.
          View in: PubMed
        10. Bennett BT, Knight KL. Cellular localization of human Rad51C and regulation of ubiquitin-mediated proteolysis of Rad51. J Cell Biochem. 2005 Dec 15; 96(6):1095-109.
          View in: PubMed
        11. Lloyd JA, McGrew DA, Knight KL. Identification of residues important for DNA binding in the full-length human Rad52 protein. J Mol Biol. 2005 Jan 14; 345(2):239-49.
          View in: PubMed
        12. Forget AL, Bennett BT, Knight KL. Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51. J Cell Biochem. 2004 Oct 15; 93(3):429-36.
          View in: PubMed
        13. McGrew DA, Knight KL. Molecular design and functional organization of the RecA protein. Crit Rev Biochem Mol Biol. 2003; 38(5):385-432.
          View in: PubMed
        14. Lloyd JA, Forget AL, Knight KL. Correlation of biochemical properties with the oligomeric state of human rad52 protein. J Biol Chem. 2002 Nov 29; 277(48):46172-8.
          View in: PubMed
        15. Logan KM, Forget AL, Verderese JP, Knight KL. ATP-mediated changes in cross-subunit interactions in the RecA protein. Biochemistry. 2001 Sep 25; 40(38):11382-9.
          View in: PubMed
        16. Kelley De Zutter J, Forget AL, Logan KM, Knight KL. Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament. Structure. 2001 Jan 10; 9(1):47-55.
          View in: PubMed
        17. Eldin S, Forget AL, Lindenmuth DM, Logan KM, Knight KL. Mutations in the N-terminal region of RecA that disrupt the stability of free protein oligomers but not RecA-DNA complexes. J Mol Biol. 2000 May 26; 299(1):91-101.
          View in: PubMed
        18. De Zutter JK, Knight KL. The hRad51 and RecA proteins show significant differences in cooperative binding to single-stranded DNA. J Mol Biol. 1999 Nov 5; 293(4):769-80.
          View in: PubMed
        19. Skiba MC, Logan KM, Knight KL. Intersubunit proximity of residues in the RecA protein as shown by engineered disulfide cross-links. Biochemistry. 1999 Sep 14; 38(37):11933-41.
          View in: PubMed
        20. Konola JT, Guzzo A, Gow JB, Walker GC, Knight KL. Differential cleavage of LexA and UmuD mediated by recA Pro67 mutants: implications for common LexA and UmuD binding sites on RecA. J Mol Biol. 1998 Feb 20; 276(2):405-15.
          View in: PubMed
        21. Kelley JA, Knight KL. Allosteric regulation of RecA protein function is mediated by Gln194. J Biol Chem. 1997 Oct 10; 272(41):25778-82.
          View in: PubMed
        22. Nastri HG, Guzzo A, Lange CS, Walker GC, Knight KL. Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site. Mol Microbiol. 1997 Sep; 25(5):967-78.
          View in: PubMed
        23. Logan KM, Skiba MC, Eldin S, Knight KL. Mutant RecA proteins which form hexamer-sized oligomers. J Mol Biol. 1997 Feb 21; 266(2):306-16.
          View in: PubMed
        24. Konola JT, Nastri HG, Logan KM, Knight KL. Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities. J Biol Chem. 1995 Apr 14; 270(15):8411-9.
          View in: PubMed
        25. Nastri HG, Knight KL. Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities. J Biol Chem. 1994 Oct 21; 269(42):26311-22.
          View in: PubMed
        26. Konola JT, Logan KM, Knight KL. Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site. J Mol Biol. 1994 Mar 18; 237(1):20-34.
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        27. Skiba MC, Knight KL. Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli. J Biol Chem. 1994 Feb 4; 269(5):3823-8.
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        28. Logan KM, Knight KL. Mutagenesis of the P-loop motif in the ATP binding site of the RecA protein from Escherichia coli. J Mol Biol. 1993 Aug 20; 232(4):1048-59.
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