Hopping Enables a DNA Repair Glycosylase To Search Both Strands and Bypass a Bound Protein

Abstract

Spontaneous DNA damage occurs throughout the genome, requiring that DNA repair enzymes search each nucleotide every cell cycle. This search is postulated to be more efficient if the enzyme can diffuse along the DNA, but our understanding of this process is incomplete. A key distinction between mechanisms of diffusion is whether the protein maintains continuous contact (sliding) or whether it undergoes microscopic dissociation (hopping). We describe a simple chemical assay to detect the ability of a DNA modifying enzyme to hop and have applied it to human alkyladenine DNA glycosylase (AAG), a monomeric enzyme that initiates repair of alkylated and deaminated purine bases. Our results indicate that AAG uses hopping to effectively search both strands of a DNA duplex in a single binding encounter. This raised the possibility that AAG might be capable of circumnavigating blocks such as tightly bound proteins. We tested this hypothesis by binding an EcoRI endonuclease dimer between two sites of DNA damage and measuring the ability of AAG to act at both damaged sites in a single binding encounter. Remarkably, AAG bypasses this roadblock in ∼50% of the binding events. We infer that AAG makes significant excursions from the surface of the DNA, allowing reorientation between strands and the bypass of a bound protein. This has important biological implications for the search for DNA damage because eukaryotic DNA is replete with proteins and only transiently accessible.The human base excision DNA repair pathway repairs ∼10,000 lesions per cell per day (1). This is a daunting task because these relatively rare lesions must be located from among ∼12,000,000,000 normal nucleotides in the genome. Almost a dozen different human DNA repair glycosylases continuously and independently search the genome for a wide variety of oxidized or alkylated bases. Once a damaged nucleotide has been located, the glycosylase catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base and create an abasic site. This abasic site is further processed to restore the correct DNA sequence using the opposing nucleotide as a template. There is considerable in vitro evidence that glycosylases use thermally-driven linear diffusion to efficiently search for sites of damage, whereby the enzyme diffuses along DNA in a non-directional manner, searching many adjacent sites within a single binding event (2-8). The biological importance of linear diffusion has been confirmed by the findings that mutants of T4 pyrimidine dimer glycosylase and EcoRV endonuclease that are deficient in linear diffusion have decreased activity in vivo (9-11).The task of locating specific sites within the genome is central to DNA repair and to many other nuclear processes such as DNA replication and transcription (12)(13)(14). Two distinct mechanisms are recognized for diffusion along DNA and they are commonly referred to as [13][14][15][16][17][18][19][20][21][22]. As illustrated in Scheme 1, sliding involves continuous contact between the protein and the D...

Structure of Escherichia coli AlkA in Complex with Undamaged DNA

“…Additionally, evidence has been presented that glycosylases undergo microscopic dissociation or "hopping" to avoid protein "obstacles" on the DNA (49). The fleeting nature of these translocation intermediates has rendered them elusive from the vantage point of structural characterization at high resolution.…”

Insights into the glycosylase search for damage from single-molecule fluorescence microscopy

“…Correlated cleavage studies have used linear DNA with blockades or breaks to determine whether a glycosylase can “hop” over the impedence. The first study looked at the enzymatic turnover of human alkyladenine DNA glycosylase (AAG) as it interacted with two lesions separated by an EcoRI binding site [28]. The presence of the EcoRI enzyme bound to the site between the lesions decreased the processivity of AAG by only 50%, indicating that some of the glycosylases were able to hop over the restriction enzyme to process the second lesion.…”

Mechanism of translocation of uracil–DNA glycosylase from Escherichia coli between distributed lesions

“…For DNA repair enzymes, the 1D diffusion mechanism was most thoroughly studied for bacteriophage T4 endonuclease V (reviewed in [9]), which even has been shown to use 1D search in vivo . Other examples of 1D search include enzymes of base excision repair ( E. coli and mammalian uracil-DNA glycosylases [10; 11; 12; 13], human alkyladenine-DNA glycosylase [14; 15], 8-oxoguanine-DNA glycosylases from E. coli [16; 17], Bacillus stearothermophilus [18] and human [17; 18], E. coli MutY DNA glycosylase [16], human abasic site endonuclease [19], and several proteins involved in direct reversal, mismatch repair, and recombination repair (reviewed in [1; 2]).…”