It may be estimated that during each day of our lives about 104
base lesions arise in each cell of our bodies (41
). Since mutations do not also arise with such alarming frequency, it must be concluded that base excision repair efficiently corrects these lesions, providing a lower limit to the efficiency by which DNA glycosylases locate these sites and initiate repair. In a genome size of ~7 × 109
bp and given the relatively high abundance of DNA repair glycosylases (~105
), it may be estimated that an individual glycosylase must survey about 70,000 bps of DNA. Of course this calculation assumes that all the base pairs of DNA are accessible to the glycosylase, which may not be true given the highly packed structure of chromatin, but it does give one measure of the magnitude of the problem.
It is of interest to ask how long the target search takes in a human nucleus? As a first approximation of the problem, one may use the Smoluchowski diffusion equation to calculate the average time for encounter of a single enzyme molecule with a target base pair in the absence of interactions
with the rest of the DNA chain (eq 1
). Equation 1
simply states that the search time (tsearch, 3D
) for diffusional encounter with a damaged base pair
increases in direct proportion to the volume of solution that must be searched (in this case the nuclear volume), and shortens with increasing rates of three-dimensional diffusion (D3
), and as the target radius (r
) increases (big targets are more likely to be encountered than small targets). Using reasonable values for D3
/s for a typical enzyme with a diameter of 5 nm, a nuclear volume of ~1011
, and a target radius of 0.34 nm for a single base pair, a search time of less than one hour may be calculated, which seems rapid enough compared to the average time for DNA replication in human cells. Although diffusion constants may be smaller in the nucleus, direct measurements indicate that this diminishment is less than 10-fold (44
)*, and the search time would still smaller than the typical dividing time of a cell. Moreover, the time that is required for target location by simple diffusion is decreased in direct proportion to the number enzyme molecules present in the nucleus (~105
), which when considered, pushes the calculated search time down to a very short time scale indeed.
However, this most simple of diffusional mechanisms becomes unsatisfactory when one considers that each enzyme molecule must locate a single damaged base pair that exists in a 30,000-fold excess of nonspecific DNA binding sites. Thus, it is 30,000 fold more probable that the enzyme will encounter undamaged DNA rather the target site. If no pathway existed for the transfer of the enzyme from a nonspecific site to a damage site, then undamaged DNA would serve as a potent competitive inhibitor of damage site repair. Fortunately, these enzymes have evolved a capacity to use nonspecific binding interactions with the DNA chain in a discrete search mode that allows efficient intramolecular transfer from an initial non-specific binding site to the site of damage (45
). As we further develop below, intramolecular transfer, even over short segment lengths, serves another important purpose beyond providing a pathway to the target site: the enzyme remains in contact with the DNA, which is a fundamental requirement if rare sites of damage are to be detected.