DNA Repair

Mismatch repair proteins correct base-pair mismatches and small insertion/deletion mismatches that arise in DNA from errors during DNA replication and recombination, or from DNA damage. If left uncorrected, these errors can be incorporated into the genetic code in the next cycle of DNA replication. Thus, defective mismatch repair results in elevated mutation rates and genome instability. In humans, an impaired or inactive mismatch repair system predisposes cells to tumor development. The striking discovery, in 1993, that mutations in mismatch repair genes are intimately linked to hereditary nonpolyposis colon cancer has stimulated great interest in the process of eukaryotic mismatch repair.

Recent studies have also implicated mismatch repair proteins in cellular processes such as mitotic and meiotic DNA recombination, transcription-coupled DNA repair, resistance to drugs and ionizing radiation, apoptosis, and possibly breast cancer. Thus, elucidating the mechanisms by which these proteins work appears key to understanding multiple pathways of DNA metabolism and how they relate to the well-being of the cell, as well as for understanding the role of mismatch repair proteins in cancer.

Knowledge of the eukaryotic mismatch repair system is rooted in seminal work performed with E. coli. Briefly, the E. coli repair system comprises: MutS protein, that recognizes a DNA mismatch, MutH, that nicks the incorrect DNA strand, and MutL, that mediates interactions between MutS and MutH. A recent study of these proteins suggests that after assembling at the mismatch, the MutS-MutL complex translocates to the nearest GATC site where MutH introduces a nick in the incorrect DNA strand. Following nicking, DNA helicase and exonuclease catalyze degradation of DNA beyond the mismatch. The resulting gap is filled in by DNA polymerase and the new strand is ligated to complete repair (Schematic of E. coli mismatch repair from Jiricny, J. (1998) "Replication errors: cha(lle)nging the genome." EMBO J 17, 6427-6436).

Our lab is interested in studying the mechanisms by which DNA defects are corrected in E. coli and S. cerevisiae. The eukaryotic mismatch repair system appears more intricate than that of E. coli. There are several homologs of MutS (MSH 1 - 6) and MutL (MLH 1 - 3, PMS1) in yeast, and even in humans. In accordance with the E. coli model, the MSH proteins appear to recognize defects in DNA base-pairs, bind MLH proteins, and signal downstream events that ultimately result in DNA repair. However, many questions need to be answered in order to understand how the eukaryotic mismatch repair system works, including:

• How do MSH proteins recognize different DNA defects (e.g., single base-pair mismatches, small or large insertion/deletion loops)?
• What is the function of MLH proteins?
• Why are there so many MutS and MutL homologs in eukarotes as compared to E. coli?
• Do these proteins translocate on DNA (before mismatch recognition to scan for defects, and/or after mismatch recognition to signal downstream repair events)?
• How do the DNA repair proteins communicate--with each other and with other DNA metabolic proteins?
• How does ATP power the activity of MSH and MLH proteins?
• Our research will utilize a combination of steady-state and pre steady-state kinetic techniques, as well as protein biochemistry and molecular biology techniques to examine:
• protein-protein and protein-nucleic acid interactions among the various mismatch repair proteins
• dynamic protein conformational changes during repair
• how the above processes are coupled to ATP binding and hydrolysis
• and structure-function properties of the repair proteins in order to elucidate the mechanism of action of the mismatch repair proteins.

DNA Replication

Another focus of research in the lab will be the mechanism of assembly of circular protein clamps on DNA. Replicative DNA polymerases from several organisms utilize a sliding clamp mechanism to catalyze rapid and processive synthesis od genomic DNA. For example, E. coli DNA polymerase III holoenzyme is bound by the b sliding clamp as it replicates DNA. b encircles DNA and forms a mobile tether for the polymerase, allowing it to extend several thousand nucleotides in a single primer-template binding event.

Since b is a closed protein ring, a clamp loader is required to open the ring and load it onto DNA for use by the polymerase. In E. coli, the multi-subunit g complex catalyzes b assembly on DNA in a reaction fueled by ATP. Briefly, on binding two-three ATP molecules, the g complex undergoes a change in conformation, that allows it to bind and open the b clamp. The ATP-bound clamp loader also binds primer-template DNA with high affinity, bringing the open clamp and DNA in close proximity to each other. Next, g complex hydrolyzes the ATP molecules one at a time, and likely couples these sequential ATPase reactions to placement of DNA within the open clamp, closure of the clamp around DNA and release of the topologically linked clamp and DNA. Ongoing rapid kinetic studies are aimed at elucidating the catalytic mechanism of how g complex uses ATP hydrolysis to complete the b assembly.

An analogous sliding clamp/clamp loader mechanism is responsible for processive DNA replication in S. cerevisiae. The circular PCNA clamp is assembled around DNA by the ATP-driven action of the five-subunit RFC complex (Replication Factor C). All five RFC subunits potentially bind and hydrolyze ATP, although it is not clear which of these ATPases are directly coupled to the work of binding PCNA, opening the ring, and placing it around DNA. Moreover, the role of each subunit in the process of clamp assembly is yet to be clearly defined. Recently, we have overexpressed and purified milligram amounts of active RFC complex from E. coli. This quantity of protein makes it possible for us to examine the catalytic mechanism of action of RFC using rapid kinetic techniques. We will study several aspects of the clamp assembly reaction, including ATP binding and hydrolysis, and corresponding changes in RFC conformation that are coupled to its interactions with PCNA and DNA, as well as PCNA opening, and closure around DNA.

In addition to their known function of PCNA loading during S. cerevisiae DNA replication, RFC proteins have also been implicated in transcription, recombination, apoptosis, and cell-cycle regulatory pathways. It is anticipated that a detailed understanding of how RFC uses ATP for clamp assembly will also yield insights into its action in these other cellular processes.

Recent Publications

Jacobs-Palmer, E. and Hingorani, M.M. (2007) The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair.  J. Mol. Biol. 366: 1087-1098. 

Coman*, M. and Chen*, S. O'Donnell, M. and Hingorani, M.M. (2007) Role of an E. coli clamp loader Tryptophan in selecting primer-template DNA for clamp assembly.  submitted to Journal of Biological Chemistry. 

(*These authors contributed equally to the research)

Antony, E., Khubchandani, S. Chen, S. and Hingorani, M.M. (2006).  “Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of S. cerevisae Msh2-Msh6 mismatch repair protein”  DNA Repair 5, 153-162.

Zito*, C.R., Antony*, E.A., Hunt, J.F., Oliver, D.B. and Hingorani, M.M. (2005) "Role of a conserved glutamate reside in the E. coli SecA ATPase mechanism". Journal of Biological Chemistry 280:14611-14619.

(*These authors contributed equally to the research)

Antony, E. and Hingorani, M.M (2004) "Asymmetric ATP binding and hydrolysis activity of the T. aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA" Biochemistry 43, 13115-13128.

Coman, Maria Magdalena, Mi Jin, Razvan Ceapa, Jeff Finkelstein, Michael O'Donnell, Brian T. Chait and Manju Hingorani (2004) "Dual Functions, Clamp Opening and Primer-Template Recognition, Define a Key Clamp Loader Subunit" J. Mol. Biol 342, 1457-1469.

Hingorani, M. M. and O’Donnell, M. (2004) “DNA Elongation.” in The Bacterial Chromosome (ed., N. Pat Higgins), ASM Press, Washington D.C.

Finkelstein, J.F., Antony, E.A., Hingorani, M.M. and O'Donnell, M. (2003) "Over-production and analysis of eukaryotic multi-protein complexes in E. coli using a dual vector strategy" Anal. Biochem 319, 78-87.

Antony, Edwin and Hingorani, Manju, M. (2003) "Mismatch Recognition-Coupled Stablization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair" Biochemistry 42, 7682-7693.

Yao, N., Coryell, L., Zhang, D., Georgescu, R.E., Finkelstein, J., Coman, M.M., Hingorani, M.M. and O'Donnell, M. (2003) "Replication Factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to Proliferating Cell Nuclear Antigen." J. Biol. Chem. 278, 50744-50753.

Ason, B., Handayani, R., Williams, C., Bertram, J., Hingorani, M., O’Donnell, M., Goodman, M. and Bloom, L.B. (2003) “Mechanism of loading the E. coli DNA polymerase III b sliding clamp on DNA” J. Biol. Chem. 278, 10033 - 10040.

Coman, M.M. and Hingorani, M.M. (2002) “On the specificity of interaction between S. cerevisiae clamp loader, RFC, and primed DNA during DNA replication.” J. Biol. Chem. 277, 47213-47224.

Jeruzalmi, D., Yurieva, O., Zhao, Y., Young, M., Stewart, J., Hingorani, M., O’Donnell, M., Kuriyan, J. (2001) “Mechanism of processivity clamp opening by the d-subunit wrench of the clamp loader complex of E. coli DNA Polymerase III.” Cell 106, 417-428.

Stewart, J., Hingorani, M.M., Kelman, Z., O’Donnell, M.E. (2001) “Mechanism of b clamp opening by the d subunit of E. coli DNA Polymerase III holoenzyme.” J. Biol. Chem. 276, 19182-19189.

Leu, F. P., Hingorani, M.M., Turner, J., O'Donnell, M. E. (2000) "The d subunit of DNA polymerase III holoenzyme serves as the sliding clamp unloader in E. coli." J. Biol. Chem. 275, 34609-34618.

Bertram, J. G., Bloom, L. B., Hingorani, M. M., Beechem, J. M., O'Donnell, M., Goodman, M. F. (2000) "Molecular Mechanism and Energetics of Clamp Assembly in Escherichia coli. The role of ATP hydrolysis when g complex loads b on DNA. J. Biol. Chem. 275, 28413-28420.

Ason, B., Bertram, J. G., Hingorani, M. M., Beechem, J. M., O'Donnell, M., Goodman, M. F. and Bloom, L. B. (1999) "A model for E. coli DNA polymerase III holoenzyme assembly at primer/template ends: DNA triggers a change in binding specificity of the g complex clamp loader." J. Biol. Chem. 275, 3006-3015.

Hingorani, M. M., Bloom, L. B., Goodman, M. F. and O'Donnell, M. (1999) "Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA." EMBO J 18, 5131-5144.

Turner, J., Hingorani, M. M., Kelman, Z. and O'Donnell, M. (1999) "The internal workings of a DNA polymerase clamp-loading machine." EMBO J 18, 771-783.

Hingorani, M. M. and O'Donnell, M. (1998) "ATP binding to the E. coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme." J. Biol. Chem. 273, 24550-24563.

Hingorani, M. M., Washington, M. T., Moore, K. C. and Patel, S. S. (1997) "The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase." Proc. Natl. Acad. Sci. USA 94, 5012-5017.

Yu, X., Hingorani, M. M., Patel, S. S. and Egelman, E. H. (1996) "DNA is bound in the central hole to one or two subunits of the T7 DNA helicase." Nature Structural Biology 3, 740-743.

Hingorani, M. M. and Patel S. S. (1996) "Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases." Biochemistry 35, 2218-2228.

Principal Investigator

Manju Hingorani

Graduate Students

Siying Chen
Hanna Semke
Jie Zhai

Postdoc:

Miho Sakato

Grant Support:

National Science Foundation