Joint Appointment in Biochemistry and Biophysics
DNA Processing Enzymes and Triplet-Repeat Instability
Many enzymes are required to replicate and repair DNA. These enzymes are generally highly specific, accurate machines that are precisely controlled in their interactions with and processing of DNA. Correct functioning allows a fine balance between accurate replication and repair of DNA on the one hand and genetic evolution on the other hand. Attenuation in specificity, accuracy or control results in genomic instability and can have disastrous results phenotypically manifested as cancer and other genetic diseases.
Our efforts to study DNA processing interactions are focused in two research areas. One area of focus is triplet-repeat instability. The human genome contains many triplet-repeat tracts that can greatly expand in size upon transmission from parent to child by a process known as dynamic mutation. Expansion of CCG trinucleotide-repeat tracts gives rise to the folate-sensitive group of fragile sites. One of these, fragile X (FRAXA), is responsible for the most common familial form of mental retardation. Expansions of CAG-repeat tracts give rise to a number of neuromuscular disorders, including myotonic dystrophy and Huntington’s disease. Our laboratory is developing model systems in vitro and in human cells to study the molecular basis for repeat expansion. Recent studies from our lab have shown the ability of DNA damage to induce massive expansions during DNA replication in vitro. Studies in progress are characterizing the intermediates in the expansion reaction and the mechanisms of expansion.
A second area of focus is the fascinating enzyme NaeI, which is both a type II restriction endonuclease and a type I topoisomerase and recombinase depending on the amino acid at position 43. We found that NaeI-43K relaxes DNA in a step-wise manner, decatenates k-DNA, recombines pBR322 in vitro, and forms a transient covalent intermediate with its DNA substrate, and yet NaeI-43K has no sequence similarity to any reported topoisomerase or recombinase. Thus, NaeI provides a new structure to study topoisomerase and recombinase activities. In addition, understanding the cleavage mechanisms of the restriction endonuclease domain is important because it occurs in some DNA repair enzymes and transposases that affect DNA stability. Our laboratory is mapping the structure-function relations within NaeI protein using biochemistry and genetics. In collaboration with Dr. Hengming Ke’s laboratory, we recently reported the 3-D structures of both NaeI protein and NaeI protein complexed with DNA. The structures, like the functions of NaeI, bridge the endonuclease and topoisomerase/recombinase protein families. Surprisingly, NaeI recognizes two copies of the same DNA sequence using widely different sets of amino acids and different protein folds located in the same polypeptide. This represents the first example of dual-mode DNA recognition by a protein. Present studies focus on the mechanisms that enable NaeI DNA endonuclease, topoisomerase, and recombinase activities in the same protein.
- The Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008 Sep 4.
- Ruggiero BL, Topal MD. Triplet repeat expansion generated by DNA slippage is suppressed by human flap endonuclease 1. J Biol Chem. 2004 May 28;279(22):23088-97. Epub 2004 Mar 22.
- Heidenfelder BL, Topal MD. Effects of sequence on repeat expansion during DNA replication. Nucleic Acids Res. 2003 Dec 15;31(24):7159-64.
- Carrick KL, Topal MD. Amino acid substitutions at position 43 of NaeI endonuclease. Evidence for changes in NaeI structure. J Biol Chem. 2003 Mar 14;278(11):9733-9. Epub 2003 Jan 8.
- Heidenfelder BL, Makhov AM, Topal MD. Hairpin formation in Friedreich’s ataxia triplet repeat expansion. J Biol Chem. 2003 Jan 24;278(4):2425-31. Epub 2002 Nov 18.
- Huai Q, Colandene JD, Topal MD, Ke H. Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement. Nat Struct Biol. 2001 Aug;8(8):665-9.
- Colandene JD, Topal MD. Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase. Biochemistry. 2000 Nov 14;39(45):13703-7.
- Huai Q, Colandene JD, Chen Y, Luo F, Zhao Y, Topal MD, Ke H. Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase. EMBO J. 2000 Jun 15;19(12):3110-8.
- Lyons-Darden T, Topal MD. Abasic sites induce triplet-repeat expansion during DNA replication in vitro. J Biol Chem. 1999 Sep 10;274(37):25975-8.