Nicholas R. Pannunzio

Non-homologous DNA end joining and alternative pathways to double-strand break repair

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Non-homologous end joining often uses microhomology: Implications for alternative end joining

Artemis and PALF (also called APLF) appear to be among the primary nucleases involved in non-homologous end joining (NHEJ) and responsible for most nucleolytic end processing in NHEJ. About 60% of NHEJ events show an alignment of the DNA ends that use 1 or 2bp of microhomology (MH) between the two DNA termini. Thus, MH is a common feature of NHEJ. For most naturally occurring human chromosomal deletions (e.g., after oxidative damage or radiation) and translocations, such as those seen in human neoplasms and as well as inherited chromosomal structural variations, MH usage occurs at a frequency that is typical of NHEJ, and does not suggest major involvement of alternative pathways that require more extensive MH. Though we mainly focus on human NHEJ at double-strand breaks, comparison on these points to other eukaryotes, primarily S. cerevisiae, is informative.

The strength of an Ig switch region is determined by its ability to drive R loop formation and its number of WGCW sites.

R loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R loop formation, and on the number of WGCW sites.

The role of G-density in switch region repeats for immunoglobulin class switch recombination

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.

Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids

BackgroundLong genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KD that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.FindingsAs our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.ConclusionAny use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.

Dissecting the Roles of Divergent and Convergent Transcription in Chromosome Instability.

The interplay of transcription, topological tension, and chromosome breakage is a subject of intense interest, but, with so many facets to the problem, it is difficult to test. Here, we vary the orientation of promoters relative to one another in a yeast system that permits sensitive detection of chromosome breaks. Interestingly, convergent transcription that would direct RNA polymerases into one another does not increase chromosome breakage. In contrast, divergent transcription that would create underwound and potentially single-stranded DNA does cause a marked increase in chromosome breakage. Furthermore, we examine the role that topoisomerases are playing in preventing genome instability at these promoters and find that Top2 is required to prevent instability at converging promoters.

Convergent BCL6 and lncRNA promoters demarcate the major breakpoint region for BCL6 translocations

To the editor: We recently published a study showing that BCL6 translocations occur at distinct sequence motifs within intron 1 of the BCL6 gene that are recognized and deaminated by activation-induced cytidine deaminase (AID).[1][1] It has been unclear, however, why these breakpoints cluster in an

RNA Polymerase Collision versus DNA Structural Distortion: Twists and Turns Can Cause Break Failure.

The twisting of DNA due to the movement of RNA polymerases is the basis of numerous classic experiments in molecular biology. Recent mouse genetic models indicate that chromosomal breakage is common at sites of transcriptional turbulence. Two key studies on this point mapped breakpoints to sites of either convergent or divergent transcription but arrived at different conclusions as to which is more detrimental and why. The issue hinges on whether DNA strand separation is the basis for the chromosomal instability or collision of RNA polymerases.

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Concept of DNA Lesion Longevity and Chromosomal Translocations

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.