Activation-induced deaminase (AID) belongs to the APOBEC family enzymes which convert cytosines in nucleic acids to uracil. AID is required for two processes essential for antibody maturation- somatic hypermutations and class-switch recombination Additionally, AID is also known to cause hypermutations in oncogenes and promote chromosome translocations that are hallmark of cancer. Among the unanswered questions regarding the role of AID in carcinogenesis is how AID selects certain genes and some loci for deamination. During the last funding period we studied the role played by transcription in the targeting process;in this proposal we will investigate how substrate selection by this enzyme affects the genetic outcomes. We will determine how the frequency, location and spectrum of genetic alterations changes when the substrate specificity of AID is altered. To accomplish this an AID- APOBEC3G hybrid with a strong preference for deaminating the last cytosine in a run of C's will be introduced in AID-/- murine B cells and the resulting hypermutations, isotype switching and chromosome translocations will be studied. The translocation of c-myc gene to one of the Ig genes is known to require AID and the junctions of such translocations promoted by mutant AID constructs will be identified and sequenced. AID will also be mutated to change its poor efficiency of converting 5-methylcytosine (mC) in DNA to thymine. Such conversions have been tied to DNA demethylation that is essential for reprogramming of embryonic cells and to translocation hotspots responsible for a number of human lymphoid malignancies. The demethylation activity of the mutants will be confirmed and AID mutants with altered preference for mC will be expressed in AID-/- B cells to determine whether the frequency, spectrum or locations of chromosome translocations are altered. These studies will create novel tools to study genetic instability promoted by AID and will answer specific questions regarding how the selection of DNA substrates by AID at nucleotide level influences its beneficial as well as harmful biological effects.

Public Health Relevance

Cancer is a disease that is caused by alterations in human DNA such as mutations and abnormal physical linking of different chromosomes (translocations). An enzyme called AID that is required for making antibodies and which helps us battle infections, has been linked to such harmful chromosome aberrations. We propose here experiments that will alter the way this enzyme interacts with DNA and determine the effects of this change on mutations and chromosome translocations using a mouse genetic model.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057200-11
Application #
8477200
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Marino, Pamela
Project Start
1998-06-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
11
Fiscal Year
2013
Total Cost
$267,012
Indirect Cost
$83,662
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Carpenter, Michael A; Rajagurubandara, Erandi; Wijesinghe, Priyanga et al. (2010) Determinants of sequence-specificity within human AID and APOBEC3G. DNA Repair (Amst) 9:579-87
Betham, Brittany; Shalhout, Sophia; Marquez, Victor E et al. (2010) Use of Drosophila deoxynucleoside kinase to study mechanism of toxicity and mutagenicity of deoxycytidine analogs in Escherichia coli. DNA Repair (Amst) 9:153-60
Canugovi, Chandrika; Samaranayake, Mala; Bhagwat, Ashok S (2009) Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase. FASEB J 23:34-44
Fix, Douglas; Canugovi, Chandrika; Bhagwat, Ashok S (2008) Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli. DNA Repair (Amst) 7:1289-97
Szczepanowski, Roman H; Carpenter, Michael A; Czapinska, Honorata et al. (2008) Central base pair flipping and discrimination by PspGI. Nucleic Acids Res 36:6109-17
Carpenter, Michael A; Bhagwat, Ashok S (2008) DNA base flipping by both members of the PspGI restriction-modification system. Nucleic Acids Res 36:5417-25
Bhagwat, Ashok S; Carpenter, Michael A; Bujnicki, Janusz M (2008) Is AID a monomer in solution? DNA Repair (Amst) 7:349-50;author reply 351-2
Roy, Todd W; Bhagwat, A S (2007) Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation. Nucleic Acids Res 35:e147
Samaranayake, Mala; Bujnicki, Janusz M; Carpenter, Michael et al. (2006) Evaluation of molecular models for the affinity maturation of antibodies: roles of cytosine deamination by AID and DNA repair. Chem Rev 106:700-19
Carpenter, Michael; Divvela, Pradeep; Pingoud, Vera et al. (2006) Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI. Nucleic Acids Res 34:3762-70

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