DNA Substrate Length and Surrounding Sequence Affect the Activation-induced Deaminase Activity at Cytidine

Yu, Kefei; Huang, Feng Ting; Lieber, Michael R.

doi:10.1074/jbc.m311616200

Cited by 179 publications

(163 citation statements)

References 33 publications

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“…Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5)(6)(7)(8)(9)(10)(11). These findings are consistent with previous experiments in which SHM is linked to transcription (12,13).…”

supporting

confidence: 83%

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Sui

Storb

2004

Proc. Natl. Acad. Sci. U.S.A.

141

149

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The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.A major advance in the study of somatic hypermutation (SHM) and class-switch recombination (CSR) has been the discovery of the activation-induced cytidine deaminase (AID) (1-3), which appears to be the long-sought SHM mutator factor and inducer of CSR (4). Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5-11). These findings are consistent with previous experiments in which SHM is linked to transcription (12, 13). However, in vivo, both DNA strands are equally mutated (14). Furthermore, in ung Ϫ/Ϫ mice, where almost all of the C or G mutations are transitions due to unrepaired AID lesions, equal targeting of both strands is confirmed (15). Single-stranded DNA would also occur in vivo as a potential AID target during DNA replication, which would explain why both DNA strands are targeted during SHM and CSR. However, experiments with a cell line that undergoes SHM in culture support the conclusion that AID can act during the G 1 phase of the cell cycle, and therefore, is not restricted to the S phase (16) (S. Gasior and U.S., unpublished data). A nonexclusive third possibility is that DNA topology (e.g., supercoiling) creates an AID-accessible conformation. In vivo, Ig genes are associated in nucleosomes with histones and other chromatin proteins. These associations, as well as the process of transcription, affect the topology of DNA. To test the possibility that supercoiled DNA as it exists in vivo may be a target for AID, we carried out cytidine deamination assays in vitro with AID purified from insect cells (5). The supercoiled target DNA was an Escherichia coli plasmid that had been manipulated to allow bacterial resistance to carbenicillin only when the initiator AUG of an ampicillin resistance (Amp r ) gene was created by AID deamination of an ACG triplet. Materials and MethodsPlasmid Construction. The kanamycin-resistance (Kan r ) gene was inserted into the SacII site in pBluescript KS(II...

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supporting

confidence: 83%

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Sui

Storb

2004

Proc. Natl. Acad. Sci. U.S.A.

141

149

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“…We have established that AID is responsible for initiating most DNA lesions that lead to human B cell translocations (3,5,6,8,21,22). In order to deaminate C to U or methyl-C to T in these translocation fragile zones, these zones must be accessible to AID (26,27). The boundary match between fragile zones and hypomethylation zones provides possible support for such a hypothesis.…”

Section: Resultsmentioning

confidence: 99%

“…These zones may remain hypomethylated at the subsequent pro-B stage of development when the DNA binding protein that protects these zones is no longer present, and that is why we detect high chromatin accessibility with no footprint of protein binding in these zones by GC methyltransferase and M.Alu mapping. AID recognizes only ssDNA as a substrate (26,27); therefore, DNA breathing gives AID the opportunity to deaminate any methyl-C in the fragile zone to a T. When this occurs, a T-G mismatch would arise when the DNA strands reanneal. The T-G mismatch can give rise to a doublestrand break if it is cut by the RAG complex or Artemis (2,8).…”

mentioning

confidence: 99%

Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

Lieber

Tsai

et al. 2015

Molecular and Cellular Biology

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Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100-to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.C hromosomal translocations are seminal events in the development of many hematologic malignancies (1). Many hematopoietic malignancies are associated with recurrent reciprocal translocations and their resulting gene fusions or amplifications, which guide diagnosis, assessment of prognosis, and treatment. One example is the BCL2-IGH translocation, t(14;18)(q32;q21), observed in 85% of follicular lymphomas (FL) and 30% of diffuse large B cell lymphomas (DLBCL). A second example is the CCND1-IGH translocation, t(11;14)(q13;q32), observed in almost all mantle cell lymphomas (MCL). Oncogenes near sites of recurrent translocation often confer a selective growth advantage (2). For unknown reasons, human chromosomal translocation breakpoints near these oncogenes frequently cluster within tight DNA zones (often only 20 to 600 bp in length) known as fragile zones (see the green starbursts in the upper portion of Fig. 1A and 2A). These zones are 100-to 1,000-fold more sensitive to breakage than the adjacent DNA regions located, for example, only 20 bp to either side of the fragile zone (2-7). Hence, the DNA sequences surrounding each oncogene are not equally sensitive to breakage. The molecular basis for such an extreme breakage propensity within such a short zone of DNA is unknown, despite our progress on elucidation of other aspects of the mechanism described below (8).We have previously reported that breakpoints of human lymphoid translocation fragile zones are not randomly distributed (Fig. 1A and 2A). Pro-B/pre-B cell-stage translocations show a breakage propensity for the CG (commonly called CpG but designated CG in this paper) DNA sequence motif within the 20 to 600-bp fragile zones (3). We have proposed a model in which activation-induced deaminase (AID) initiates double-strand breaks (DSBs) by acting at the methylated form of these CG sites in single-stranded DNA (ssDNA) to deaminate the 5-methylcytosine to yield a T-G mismatch (3). The r...

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“…One possible mechanism for such a coupling to occur is through replication protein A (RPA), which has been shown to bind both phosphorylated AID (Chaudhuri et al, 2004) and Ung (Otterlei et al, 1999). In an in vitro experiment, half of AGCA in a single-stranded oligonucleotide was deaminated by AID and deglycosylated by Ung within 5 minutes (Yu et al, 2004), suggesting that Ung, if at a high local concentration, as effected by either a high protein level or a high k cat /K m ratio, would be able to process most of dU in ssDNA.…”

Section: Abasic Sites Generated By Ung Stall the Replication Forkmentioning

confidence: 99%

“…SHM preferential targets the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) mutational hotspot, which contains the preferred AID deamination motif WRC (Petersen-Mahrt and Neuberger, 2003;Pham et al, 2003;Yu et al, 2004). dU is a non-bulky DNA lesion that, if not repaired, can pair with dA without blocking the DNA replication fork, thereby giving rise to dG → dA and dC → dT transitions.…”

Section: Introductionmentioning

confidence: 99%

DNA Replication to Aid Somatic Hypermutation

Zan

Pál

et al.

Advances in Experimental Medicine and Biology

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DNA Substrate Length and Surrounding Sequence Affect the Activation-induced Deaminase Activity at Cytidine

Cited by 179 publications

References 33 publications

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

DNA Replication to Aid Somatic Hypermutation

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