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The Path to DNA Repair

Although scientists have made monumental strides in deciphering the structure of DNA binding proteins and understanding their DNA interactions, there is still much to learn about the function of these biomolecules. Many questions remain about the mechanisms of DNA replication, recombination, and repair, particularly how exposure to environmental toxicants affects these processes.

Samuel Wilson Source: Samuel Wilson

Samuel Wilson, deputy director of the NIEHS, is also head of the Nucleic Acid Enzymology Group in the NIEHS Laboratory of Structural Biology. This group is interested in mammalian DNA repair, specifically how enzymes fill in gaps at places where DNA has been damaged.

A main focus of the group is understanding the mechanism and role of a DNA repair pathway called base excision repair (BER), as well as that of a central BER protein--DNA polymerase ß--which is involved in repair DNA synthesis. Many of the group's experiments explore the role that BER plays in the repair of DNA damage from endogenous and environmental sources and how this role may influence carcinogenesis and other disease processes.

DNA Repair: The Basics

The BER pathway repairs DNA damage at sites where nucleotides have lost their purine or pyrimidine bases. If a nucleotide base is damaged by either exogenous or endogenous agents, the base can be lost by spontaneous or enzymatic removal, resulting in an apurinic/apyrimidinic (AP), or abasic, site. In 1986, Wilson and colleagues at the National Cancer Institute in Bethesda, Maryland, were the first to clone the DNA polymerase ß gene, one of several known mammalian DNA polymerases involved in nuclear transactions. The group found that expression of the DNA polymerase ß gene is a regulated, adaptive process in response to environmental stress in mammalian cells. To better understand the cellular roles of DNA polymerase ß, Wilson's laboratory collaborated with the laboratory of Klaus Rajewsky of the Institute of Genetics at the University of Cologne to construct DNA polymerase ß knock-out cell lines from a transgenic mouse strain. These cell lines, which lacked DNA polymerase ß, were deficient in BER and were hypersensitive to simple alkylating agents that target DNA. The researchers discovered that DNA polymerase ß is responsible for identifying and inserting the correct nucleotide during repair of the damaged DNA strand.

These knock-out studies have helped to establish the structural and biochemical background for polymerase studies. Wilson and colleagues can now examine the structural and functional roles of DNA polymerase ß in the cell and determine how the cell's sensitivity to chemotherapeutic or environmental agents may be affected.

In 1996, just before Wilson moved his lab from the Sealy Center for Molecular Science at the University of Texas Medical Branch in Galveston, Texas, to the NIEHS, he and colleagues contributed to the identification of a second role played by DNA polymerase ß in BER. In addition to synthesizing replacement nucleotides, DNA polymerase ß also prepares the AP site for the completion of BER. A nucleotide consists of three parts: a base moiety, a sugar moiety, and a phosphate moiety. BER is initiated by removal of the damaged base. This leaves a sugar-phosphate moiety, which forms part of the backbone of one strand of the DNA. To remove the sugar-phosphate moiety at the AP site, two incisions must be made to "cut" it out. DNA polymerase ß performs the second incision, which releases the sugar-phosphate group. The resulting one-nucleotide gap serves as a template for DNA polymerase ß-dependent insertion of the new nucleotide. Once the correct nucleotide is incorporated, the resulting nick in the DNA can be sealed by a DNA ligase. If DNA polymerase ß does not remove the sugar-phosphate, the nick will not be sealed by ligase. Wilson's group has collaborated with NIEHS researcher Kenneth Tomer's lab to identify the amino acid residue in DNA polymerase ß that assists removal of the sugar-phosphate moiety, thus allowing scientists to propose a chemical mechanism for the enzymatic reaction.

Wilson and colleagues have demonstrated that DNA polymerase ß and DNA ligase I form a complex, suggesting that a complex between the proteins probably exists in the cell. The observation that DNA ligase I and DNA polymerase ß physically interact and that they are involved in BER suggests that they work together rather than independently. This is consistent with the observation that DNA polymerase ß also interacts with other BER proteins, and suggests the existence of a multiprotein BER complex, or "repairosome." Scientists have not yet determined how such repairosomes influence overall BER.

Quality Control Mechanisms

Many questions remain about how DNA polymerase ß and other polymerases select the correct nucleotide for insertion from a pool of structurally similar molecules. Unaltered wild-type forms of polymerases rarely make insertion errors, but mutant forms of the polymerase can become highly error-prone. In collaboration with Thomas Kunkel, chief of the Laboratory of Structural Biology, the researchers discovered that DNA polymerase ß sometimes inserts an incorrect nucleotide. This is especially true when protein interactions with the DNA strand that directs synthesis are altered by site-specific modification of the DNA polymerase ß gene. The resulting mutant DNA polymerase ß gene is overexpressed in Escherichia coli cells to produce a recombinant protein that can be systematically studied in vitro.

William Beard, a senior research fellow in Wilson's lab, says that he and his colleagues are interested in how nucleotide insertion mistakes occur and are corrected. In a mammalian cell, it has been estimated that DNA polymerase ß is involved in the repair synthesis of up to 1,000,000 nucleotides per day. Thus, if DNA polymerase ß makes only 1 mistake per 10,000 nucleotides synthesized, a typical mammalian cell may accumulate as many as 100 mutations per day due to BER. The ability of a DNA polymerase to select the correct nucleotide represents one mechanism to avoid mistakes. While DNA polymerase ß selects the correct nucleotides nearly as well as other DNA polymerases, it still makes enough mistakes to suggest that additional mechanisms exist to improve the overall fidelity (or ability to insert the correct nucleotide) of BER. For example, DNA polymerase ß may make fewer mistakes when it is in complex with other BER enzymes (as in a repairosome). The researchers are interested in finding additional mechanisms to improve the fidelity of BER.

William Beard Photo credit: Steve McCaw/Image Associates, Inc.

Wilson's group will continue to study DNA polymerase ß to determine what other roles it may play in the cell. They will also continue to investigate BER, which is not a single, simple pathway, but rather a collection of many alternate pathways in mammalian cells.

Pursuing Alternate Routes

Many cancer researchers are looking for alterations in the DNA polymerase ß gene that may give rise to altered polymerase molecules or BER pathways in cells. With a better understanding of the regulation and roles of DNA repair pathways, it may be possible for researchers to consider manipulating these pathways to modulate repair in a cell-specific manner.

In 1994, Wilson's group, along with Joseph Kraut, professor emeritus in the Department of Chemistry at the University of California at San Diego, was the first to determine the structure of a DNA polymerase ternary complex, in which a polymerase binds with DNA and a correct incoming nucleotide. The structure of this catalytic polymerase complex showed scientists that significant conformational changes, or molecular structure movements, occur upon binding the correct nucleotide. The structure has been a model for Wilson's group and others to examine the functional significance of polymerase-DNA interactions and how these interactions may be modulated by the structure of DNA.

DNA doctor. The Nucleic Acid Enzymology Group has modeled the mechanism by which DNA polymerase ß (blue) identifies and inserts the correct nucleotide (yellow) into damaged strands of DNA (red).
Photo credit: William Beard

When DNA polymerase ß binds DNA and the correct nucleotide for incorporation into the growing DNA strand, all three molecules undergo structural changes. Some or all of these structural changes affect the probability of binding and inserting the correct nucleotide. The researchers can examine the influence of these structural transitions by using modified DNA polymerase ß in the form of mutant enzymes created by site-directed mutagenesis, DNA of different structures (for example, damaged DNA or different DNA sequences), or modified nucleotides.

In a separate but related area, Wilson's research on HIV-1 reverse transcripterase (RT), the acquired immunodeficiency syndrome DNA polymerase enzyme, led to his proposal of a kinetic scheme for this replicative enzyme. A kinetic scheme is a quantitative diagram of the steps that occur during DNA synthesis. These steps constitute substrate and product binding and release, as well as chemistry.

In collaboration with the Kunkel laboratory, Wilson's group has demonstrated a strong correlation between DNA binding affinity and mistakes made by RT that result in the loss or addition of a nucleotide (referred to as frameshift fidelity). Lower DNA binding affinity is associated with lower frameshift fidelity. Frameshift fidelity and DNA binding affinity are modulated by a group of conserved RT residues that interact in a face of the structural DNA double helix that is known as the DNA minor groove. This group of conserved RT residues are collectively referred to as the "minor groove binding track." As in studies of DNA polymerase ß, the Wilson and Kunkel groups are interested in the roles that both DNA and protein structures have in HIV-1 RT function. By comparing similar studies of DNA polymerase ß and HIV-1 RT, they can identify characteristics unique to each polymerase. Because HIV-1 RT has a different structure than DNA polymerase ß, the researchers expect to find important functional differences between these two polymerases, such as how often they make mistakes, the types of mistakes made, and where the mistakes are made in relation to the sequence of the DNA in the template and product strands.

The ultimate goal is to understand the molecular events that lead to efficient polymerization with high fidelity and how environmental agents that target DNA influence such events. In addition, by comparing the behavior of several different polymerases, researchers can determine whether there are general strategies that are employed by DNA polymerases to accomplish DNA synthesis with high fidelity or whether each polymerase utilizes unique mechanisms. A fundamental understanding of these mechanisms is crucial because the accumulation of genomic mutations may be key to genetic susceptibility to disease and aging.

Brandy E. Fisher

Last Updated: July 19, 1999