Concept of DNA Lesion Longevity and Chromosomal Translocations

Abstract

A subset of chromosomal translocations related to B cell malignancy in human patients arises due to DNA breaks occurring within defined 20-600 base pair (bp) zones. Several factors influence the breakage rate at these sites including transcription, DNA sequence, and topological tension. These factors favor non-B DNA structures that permit formation of transient single-stranded DNA (ssDNA), making the DNA more vulnerable to agents such as the enzyme activation-induced cytidine deaminase (AID) and reactive oxygen species (ROS). Certain DNA lesions created during the ssDNA state persist after the DNA resumes its normal duplex structure. We propose that factors favoring both formation of transient ssDNA and persistent DNA lesions are key in determining the DNA breakage mechanism.

Figure 1. Fragile Zones in Human Lymphoid Chromosomal Translocations

Schematics of the BCL2 break cluster regions on chromosome 18, the BCL1 breakpoint region (which is downstream of the cyclin D1 (CCND1) gene) on chromosome 11, Exon 13 of E2A (also known as TCF3) on chromosome 19, and the CRLF2 breakpoint region upstream of the CRLF2 gene on the X chromosome illustrate the accumulation of breakpoints within the various regions. Breakpoint junctions sequenced from human patients demonstrate that DSBs anywhere within these regions can result in chromosomal translocations relevant for various B cell malignancies (red vertical lines represent a mapped breakpoint), within these regions, however, are regions where breakpoints appear to cluster in a non-random fashion (red starbursts). In the BCL2 region (top), relative proportions of breakpoints at the BCL2 major breakpoint region (MBR), intermediate cluster region (ICR), and minor cluster region (MCR) are shown. The MBR is located in the third exon of the BCL2 gene within the 3′ untranslated region (UTR), while the ICR and MCR are further downstream from the translated region. The 175 bp MBR, 105 bp ICR, and 561 bp MCR account for about 50%, 13%, and 5% of the BCL2 translocation breakpoints. Within the BCL1 region, the major translocation cluster (MTC) is located about 110 kb from the CCND1 gene. The 150 bp MTC contains about 30% of breakpoints, whereas the remaining 70% of events are distributed widely over the surrounding 340 kb as recently mapped and sequenced [33]. In the E2A cluster, which occurs in intron 13 of the E2A gene [34], 75% of breakpoints occur in the 23 bp E2A cluster, while the surrounding 3 kb only account for 25%. The CRLF2 cluster lies upstream of the CRLF2 gene with 32% of mapped break in the 25 kb region occurring within a 311 bp cluster. These sites of breakpoint accumulation that range from 23 to 561 bp are termed ‘fragile zones’ and every CG sequence motif in each of these fragile zones is a hotspot for human translocation [2, 33].

Figure 2. Sequence-Level View of ssDNA Formation via a Slipped-Strand Structure Leading to an AID-Induced Persistent Lesion

Transcription or increased torsional stress in C-string sequences that display a B/A intermediate conformation can lead to transient strand slippage at direct repeats and generate ssDNA on each strand of DNA. While these transient slippage events may last only milliseconds or less before returning to a duplex conformation, AID can act to deaminate either C’s or ^meC’s within preferred single-stranded target sequences (WRC). The T’s generated from deamination of ^meC’s are removed more slowly than U’s and are thus more long-lived lesions. After the DNA resumes its duplex conformation, the resulting T:G mismatch is vulnerable to DNA repair enzymes, such as activated Artemis, that may convert these to DSBs. For chromosomal translocations that occur in human B cells, Artemis is activated during DSB repair at chromosome 14 (e.g., during V(D)J recombination or Ig class switch recombination). Failure to efficiently repair the chromosome 14 break coupled with simultaneous generation of DSB at a fragile zone can lead to events that favor chromosomal translocations.

Figure 3, Key Figure. Model of DSB Formation at Human Fragile Zones

Fragile zones are prone to forming transient, non-B DNA structures based on biochemical [3, 4], enzymatic [2, 5, 25, 33], and genetic [5] data. Formation of ssDNA in the non-B state is enhanced by processes that separate the DNA strands (i.e., transcription) and increased torsional stress. The non-B structures (such as the slipped-strand structure depicted here) are short-lived, but, while formed, the ssDNA is vulnerable to damage. AID can attack ssDNA in human B cells and reactive oxygen species (ROS) can more easily oxidize DNA not protected within a DNA duplex. Oxidized bases generated by ROS and T:G mismatches generated by AID deamination of 5-methylcytosine represent long-lived, persistent lesions (relative to the efficient processing of uracil by uracil glycosylase) that, upon collapse of the non-B structure back to duplex DNA, create distortions recognized by activated Artemis nuclease and are cleaved. When the persistent lesions are adjacent on opposite strands of the DNA duplex, this process can result in a DSB.