Page 33 - 2014 Printable Abstract Book
P. 33
(S201) How Do DNA Glycosylases Locate Radiation-Damaged DNA Bases? Susan Wallace, University of
Vermont, Burlington, VT
The base-excision repair pathway is responsible for repairing the vast majority of free radical-
induced DNA damages. The first step of this pathway is catalyzed by the DNA glycosylase enzymes that
are responsible for both locating and removing DNA base lesions. Since these lesions often differ only
slightly from their normal counterparts and are present in a sea of undamaged bases, the recognition
process undertaken by the DNA glycosylases is the most difficult step. In order to examine the lesion
search process, we used a single-molecule TIRF assay to image quantum dot-labeled glycosylases
interacting with lambda DNA molecules suspended between silica beads. Bacterial Nth, Fpg and Nei,
members of two structural families, the Nth superfamily and the Fpg/Nei family, exhibit a similar diffusive
search mechanism, a continuum of motion in keeping with rotational diffusion along the DNA molecule
ranging from very slow to faster diffusion. Structural studies had shown three amino acids to be inserted
into the DNA helix after the damaged base was flipped out through the major groove into the glycoslyase’s
binding pocket. This amino acid triad was predicted to aid in flipping out the damaged base and to stabilize
the DNA duplex. When we mutated one of these residues to an alanine, the slow diffusive behavior
observed with the wild type glycosylases was no longer present suggesting that this wedge amino acid
was involved in the damage search. Furthermore, when we examined the behavior of these same
glycosylases on damage-containing DNA, we observed the glycosylases to stop upon encountering a
damage. The similarities and differences between the search behaviors of the bacterial glycosylases and
the behaviors of human OGG1, APE1 and MUTYH will also be discussed. Taken together, our data show
that glycosylases use facilitated diffusion to rotate around the DNA molecule and employ a wedge residue
to interrogate the DNA for damage. When the glycosylase locates a damage, it stops to remove it. This
work was supported by NIH grant P01 CA098993.
(S202) Ape1 has different effects on mammalian cell sensitivities to low and high linear energy transfer
1
1
1
1
1
ionizing radiation. Hongyan Wang ; Xiang Wang ; Guangnan Chen ; Xiangming Zhang ; Xiaobing Tang ;
1
2
1
1
1
Dongkyoo Park ; Francis A. Cucinotta ; Xingming Deng ; William S. Dynan ; Paul W. Doetsch ; Ya Wang, 1
1
Emory University, Atlanta, GA and University of Nevada, Las Vegas, NV
2
High linear energy transfer (LET) radiation kills more cells than low-LET radiation at the same dose.
The ratio is termed relative biological effectiveness (RBE), mainly because high-LET-induced DNA damage
is more difficult to repair. Previously, our group and others demonstrated that the RBE is involved in high-
LET radiation to interfere with non-homologous end-joining (NHEJ) but not homologous recombination
repair (HRR) when compared with low-LET radiation. We believe that the effect is attributable, in part, to
small DNA fragments (< 40 bp) directly produced by high-LET radiation, which affects Ku protein from
efficiently binding to the two ends of one such fragment at the same time, therefore reducing the NHEJ
efficiency. Here we demonstrate that Ape1, an enzyme required for processing AP (apurinic/apyrimidinic,
also known as abasic) sites, is also involved in the generation of small DNA double strand break (DSB)
fragments in high-LET irradiated cells since high-LET radiation induces more clustered DNA damage than
low-LET radiation. During the repair of the radiation-induced base damage located at the cluster DNA
damage, Ape1 digests the Ogg1 (DNA glycosylase)-induced AP sites to generate single strand breaks (SSBs)
nearby, which forms DSBs, particularly small DNA fragments. We also demonstrate that the 40 bp DNA
fragments that affect the efficiency of Ku binding to the two ends of one fragment at the same time do
31 | P a g e
Vermont, Burlington, VT
The base-excision repair pathway is responsible for repairing the vast majority of free radical-
induced DNA damages. The first step of this pathway is catalyzed by the DNA glycosylase enzymes that
are responsible for both locating and removing DNA base lesions. Since these lesions often differ only
slightly from their normal counterparts and are present in a sea of undamaged bases, the recognition
process undertaken by the DNA glycosylases is the most difficult step. In order to examine the lesion
search process, we used a single-molecule TIRF assay to image quantum dot-labeled glycosylases
interacting with lambda DNA molecules suspended between silica beads. Bacterial Nth, Fpg and Nei,
members of two structural families, the Nth superfamily and the Fpg/Nei family, exhibit a similar diffusive
search mechanism, a continuum of motion in keeping with rotational diffusion along the DNA molecule
ranging from very slow to faster diffusion. Structural studies had shown three amino acids to be inserted
into the DNA helix after the damaged base was flipped out through the major groove into the glycoslyase’s
binding pocket. This amino acid triad was predicted to aid in flipping out the damaged base and to stabilize
the DNA duplex. When we mutated one of these residues to an alanine, the slow diffusive behavior
observed with the wild type glycosylases was no longer present suggesting that this wedge amino acid
was involved in the damage search. Furthermore, when we examined the behavior of these same
glycosylases on damage-containing DNA, we observed the glycosylases to stop upon encountering a
damage. The similarities and differences between the search behaviors of the bacterial glycosylases and
the behaviors of human OGG1, APE1 and MUTYH will also be discussed. Taken together, our data show
that glycosylases use facilitated diffusion to rotate around the DNA molecule and employ a wedge residue
to interrogate the DNA for damage. When the glycosylase locates a damage, it stops to remove it. This
work was supported by NIH grant P01 CA098993.
(S202) Ape1 has different effects on mammalian cell sensitivities to low and high linear energy transfer
1
1
1
1
1
ionizing radiation. Hongyan Wang ; Xiang Wang ; Guangnan Chen ; Xiangming Zhang ; Xiaobing Tang ;
1
2
1
1
1
Dongkyoo Park ; Francis A. Cucinotta ; Xingming Deng ; William S. Dynan ; Paul W. Doetsch ; Ya Wang, 1
1
Emory University, Atlanta, GA and University of Nevada, Las Vegas, NV
2
High linear energy transfer (LET) radiation kills more cells than low-LET radiation at the same dose.
The ratio is termed relative biological effectiveness (RBE), mainly because high-LET-induced DNA damage
is more difficult to repair. Previously, our group and others demonstrated that the RBE is involved in high-
LET radiation to interfere with non-homologous end-joining (NHEJ) but not homologous recombination
repair (HRR) when compared with low-LET radiation. We believe that the effect is attributable, in part, to
small DNA fragments (< 40 bp) directly produced by high-LET radiation, which affects Ku protein from
efficiently binding to the two ends of one such fragment at the same time, therefore reducing the NHEJ
efficiency. Here we demonstrate that Ape1, an enzyme required for processing AP (apurinic/apyrimidinic,
also known as abasic) sites, is also involved in the generation of small DNA double strand break (DSB)
fragments in high-LET irradiated cells since high-LET radiation induces more clustered DNA damage than
low-LET radiation. During the repair of the radiation-induced base damage located at the cluster DNA
damage, Ape1 digests the Ogg1 (DNA glycosylase)-induced AP sites to generate single strand breaks (SSBs)
nearby, which forms DSBs, particularly small DNA fragments. We also demonstrate that the 40 bp DNA
fragments that affect the efficiency of Ku binding to the two ends of one fragment at the same time do
31 | P a g e
