Fine-structure analysis of activation-induced deaminase accessibility to class switch region R-loops.
Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.
Yu
Kefei
K
Department of Biochemistry and Molecular Biology, USC Norris Cancer Ctr., Rm. 5428, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90033, USA.
Roy
Deepankar
D
Bayramyan
Melina
M
Haworth
Ian S
IS
Lieber
Michael R
MR
D000818:Animals; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D051379:Mice; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014764:Viral Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004277:DNA, Single-Stranded; D014764:Viral Proteins; D003562:Cytidine; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase