LBNL Masthead A-Z IndexBerkeley Lab mastheadU.S. Department of Energy logoPhone BookJobsSearch
Protein Bridges DNA Base and Nucleotide Excision Repair Pathways Print

 

Alkyltransferase proteins (AGT) protect cells from the biological effects of DNA damage caused by the addition of alkyl groups (alkylation). Alkyltransferase-like proteins (ATLs) can do the same, but they lack the reactive cysteine residue that allows the alkyltransferase function, and the mechanism for cell protection has remained unknown. To address this mystery, a British–American team lead by researchers at the Scripps Research Institute recently applied a combination of x-ray structural, biochemical, and genetic studies to ATLs in the yeast Schizosaccharomyces pombe without and with damaged DNA. By showing how a process called non-enzymatic nucleotide flipping activates ATL-initiated DNA repair, their results may improve our understanding of genomic integrity and responses to DNA damage relevant to pathogens and cancer development.

DNA Repair Switch

A process similar in spirit to that of a railroad switch operates in cells when it is necessary to repair DNA damaged by normal metabolic processes or environmental agents, including cancer drugs. In this case, the switch is at the junction where a choice must be made between two essential DNA repair pathways that function to keep DNA healthy and without defects. In these two pathways, nucleotide excision repair and base repair, respectively, either a DNA structural unit (the nucleotide) or just the part of a nucleotide that determines the genetic code (the base) are removed from the damaged DNA and replaced. Proteins called alkyltransferase-like proteins (ATLs) protect cells against certain kinds of damage; however, the mechanism for choosing the DNA repair pathway has been a mystery.

A combination of structural, biochemical and genetics results obtained by Tubbs et al. has now shown how ATLs bridge the two DNA repair pathways that were previously thought to function independently from each other, and the key is a process dubbed “non-enzymatic DNA nucleotide flipping” that steers repairs into the nucleotide excision pathway. If ATLs are eventually found in humans, these results could significantly aid the development of more effective cancer treatments by showing how tumor cells can resist chemotherapy drugs that work by damaging their DNA. For example, because the ATLs aid DNA repair, the effects of the drug could be reversed, resulting in failure of the cancer treatment.

Cartoon showing ATL as bridge between base repair and nucleotide excision rrepair, two DNA repair pathways that were previously thought to function independently from each other. If ATLs are eventually found in humans, these results could significantly aid the development of more effective cancer treatments. [Figure courtesy of J.L. Tubbs, Scripps Research Institute.]

The research team started with diffraction data collected at the ALS (SIBYLS Beamline 12.3.1) and SSRL to determine structures for ATL (Atl1) without damage and with either endogenous or cigarette-smoke-derived damage. These structures, the first for any ATL, show that although Atl1 looks similar to the parts of alkyltransferase (AGT) where DNA binding and alkyl transfer occur, it has certain distinguishing features. Like AGT, Atl1 flips (transfers) a nucleotide from the DNA double helix to its active site to access damaged nucleotides. But unlike AGT and most other known DNA nucleotide-flipping proteins, this flipping is not connected to any type of enzymatic activity or catalysis. In addition, Atl1 has a larger binding pocket to accommodate a wider variety of damaged DNA nucleotides, including those that are too big to fit the AGT binding pocket, and Atl1 creates a bigger bend in DNA than AGT upon binding DNA.

Crystal structure for Atl1 (magenta) bound to DNA containing alkylation damage associated with O6-methylguanine (yellow). The ATL binding site (center, magenta) is drawn as a ball-and-stick representation.

Typically, there are two general DNA repair mechanisms for DNA alkylation damage, direct damage reversal and base excision repair (BER). For example, base alkylation damage is repaired by direct damage reversal proteins like AGT or by lesion-specific DNA enzymes (glycosylases) that initiate the BER pathway by excising bases. In contrast, there is another, more versatile DNA repair pathway called nucleotide excision repair (NER) that works by removing bulkier DNA helix-distorting lesions by excising a DNA patch containing a damaged base. Generally, NER poorly recognizes the kinds of alkylation damage repaired by alkyltransferases and is therefore not usually expected to be active in these cases.

Through a series of genetic and biochemical experiments, however, the researchers unexpectedly discovered that Atl1 function is linked to the NER pathway of DNA repair after all. It turns out that ATL bridges two DNA repair pathways (base repair and nucleotide excision repair) that were previously thought to function independently of each other. The researchers' combined results reveal a general mechanism in which ATL binds weakly distorting lesions (base damage) in a manner comparable to AGT and BER glycosylases and recruits NER-associated proteins by sculpting the weakly distorting alkylation damage into a bulky lesion that is channeled into the NER pathway.

By mapping conservation of amino acid sequences between their ATL and sequences in other ATLs, the researchers realized that their structures could also characterize the other ATLs. Analysis of lesion-binding site conservation based on the new structures led to identification of new ATLs in ancestral archaea (unicellular microorganisms without nuclei or organelles) as well as the first ATL discovered in any multicellular organism, a sea anemone. Together with already known ATLs, this discovery indicates that ATL interactions are ancestral to present-day repair pathways in all domains of life. Therefore, although no ATL has yet been discovered in humans, it is likely that an ATL or similar protein exists in humans, thereby opening the door to improved cancer chemotherapy based on alkylating agents and repair pathway control.

 



Research conducted by J.L. Tubbs, A.S. Arvai, and M.D. Kroeger (The Scripps Research Institute); S. Kanugula and A.E. Pegg (Pennsylvania State University); V. Latypov, A. Butt, A. Marriott, A.J. Watson, B. Verbeek, G. McGown, M. Thorncroft, and G.P. Margison (University of Manchester, UK); M. Melikishvili, M.G. Fried (University of Kentucky); R. Kraehenbuehl and O. Fleck (Bangor University, UK); M.F. Santibanez-Koref (Newcastle University, UK); C. Millington and D.M. Williams (University of Sheffield, UK); L.A. Peterson (University of Minnesota); and J.A. Tainer (The Scripps Research Institute and Berkeley Lab).

Research funding: National Institutes of Heath, The Skaggs Institute for Chemical Biology, North West Cancer Research Fund, Cancer Research-UK and CHEMORES. Operation of the ALS and of SSRL is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: J.L. Tubbs, V. Latypov, S. Kanugula, A. Butt, M. Melikishvili, R. Kraehenbuehl, O. Fleck, A. Marriott, A.J. Watson, B. Verbeek, G. McGown, M. Thorncroft, M.F. Santibanez-Koref, C. Millington, A.S. Arvai, M.D. Kroeger, L.A. Peterson, D.M. Williams, M.G. Fried, G.P. Margison, A.E. Pegg, and J.A. Tainer, "Flipping of alkylated DNA damage bridges base and nucleotide excision repair." Nature 459, 808 (2009).

 

ALSNews Vol. 303