Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

David Rueda

Second Advisor

Ashok Bhagwat

Abstract

SINGLE-STRANDED DNA SCANNING AND DEAMINATION WITH SINGLE-MOLECULE RESOLUTION

by

GAYAN SENAVIRATHNE

December 2013

Advisor: Dr. David Rueda & Dr.Ashok Bhagwat

Major: Chemistry (Analytical)

Degree: Doctor of Philosophy

APOBEC3G (Apo3G) and Activation-Induced cytidine Deaminase (AID) are the most notable members of APOBEC enzymes, a family of cytidine (C) deaminases with crucial functions in the immune system. In T cells, Apo3G deaminates C in viral cDNA to halt the replication of HIV 1 strains lacking viral infectivity factor (vif). In B cells, AID is required for the diversification of antibodies through initiation of somatic hypermutation (SHM), and class switch recombination (CSR), by the C deaminations in immunoglobulin genes. Activity of these enzymes requires scanning of single-stranded (ss) DNA substrates to locate deamination motifs and stochastically deaminate them to create mutational diversity.

Here we use single-molecule Förster resonance energy transfer (smFRET) to study ssDNA scanning and deamination by Apo3G and AID. Consistent with previous ensemble measurements, single-molecule trajectories exhibit short binders (<25 s) and long binders (25 s - 10 min), which are heterogeneously distributed for Apo3G and homogenously distributed for AID. Long binding trajectories reveal processive scanning of >70 nt ssDNA by both enzymes randomly, and bidirectionally with similar (~1 s-1) scanning rates. Both enzymes scan and crease ssDNA in deamination motif-dependent manner, where longer `dwelling' in scanning is observed around favorable deamination motifs located near 5' end of the DNA for Apo3G and without positional preference for AID. The positional presence of Apo3G `dwelling' may explain the asymmetric catalysis observed in ensemble experiments, on linear ssDNA, which is also confirmed by our smFRET deamination measurements based on the binding of Pfu polymerase to uracils formed upon C deamination by Apo3G. Scanning of Apo3G may consist of sliding and hopping/jumping as shown by its large salt dependency, whereas AID primarily uses sliding to scan the ssDNA, and is not sensitive to the changes in salt concentration.

We developed an smFRET assay to visualize AID scanning, coupled to DNA transcription. Our data show that AID can follow an active RNA polymerase directionally and processively with speeds >100 bp*s-1. However, transcription stalling leads to bidirectional scanning in the transcription bubble, which in turn, provides AID the necessary time window to carryout deaminations.

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