Biology Reference
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interactions between the glycosylase and the DNA molecule. This model has
been suggested by structural studies
64,65
and by a recent single-molecule
study.
99
Two groups have attempted to address the question of how glycosylases
search for a lesion using single-molecule approaches.
96,97,99
In the first study,
96
human OGG1 (oxoguanine DNA glycosylase, a member of the HhH super-
family) labeled with Cy3 was observed to undergo one-dimensional sliding
along DNA that was stretched by shear flow. A similar diffusive motion was
observed with BstFpg. These same authors showed that the one-dimensional
diffusion constants measured were consistent with the glycosylases diffusing
along the DNA helix in a rotational manner.
97
In a recent study from our
laboratory,
99
quantum dot-labeled
E. coli
Fpg, Nei, and Nth were imaged in
the absence of flow. In this study, the glycosylases were shown to diffuse along
the DNA with a broad distribution of rates that ranged over two orders of
magnitude. This broad distribution was common to all three glycosylases,
suggesting that both the Fpg/Nei family and HhH superfamily scan using a
similar mechanism. When the diffusive behavior was analyzed further, the
three glycosylases were shown to exhibit a continuum of motion that was in
keeping with rotational diffusion along the DNA molecule and that ranged
from a slow, subdiffusive to a faster, unrestricted diffusive behavior.
As described earlier, members of the Fpg/Nei family of DNA glycosylases
have three void-filling residues that are inserted into the DNA helix and aid in
flipping out the damaged base in addition to stabilizing theDNA helix.
55,56,59,64-67
The HhH superfamily uses a similar mechanism.
100,101
Interestingly, a crystal
structure of BstFpg crosslinked to undamaged DNA revealed that one of these
void-filling residues, a phenylalanine, was found to be wedged into the helix,
occupying a position analogous to its position in the Fpg complex bound to
damage-containing DNA
64
(
Fig. 2
A). These data, together with kinetics
data,
102,103
suggest that the phenylalanine may be acting as a wedge that scans
for deformability of the base pair such as in the sugar pucker. Interestingly,
when the corresponding
E. coli
wedge residue Phe111 was mutated to an
alanine, there was a significant increase in the mean diffusion constant com-
pared to the wild-type protein.
99
Moreover, the diffusive properties character-
istic of wild type were altered, that is, the slow, subdiffusive population
of glycosylases was selectively lost. Similar results were observed when the
analogous residues in Nei and Nth were mutated (Dunn
et al.
, unpublished
observations), suggesting that the slow subdiffusive glycosylases are those in-
terrogating the DNA for damages. Taken together, the data support the idea that
the Fpg/Nei family of DNA glycosylases diffuse one-dimensionally along the
DNA molecule with diffusion constants that are consistent with rotation around
the DNA molecule, presumably in the minor groove where they bind. It also
appears that at least part of the glycosylase search mechanism may be
