Nancy Maizels

The Bloom’s Syndrome Helicase Unwinds G4 DNA

BLM, the gene that is defective in Bloom’s syndrome, encodes a protein homologous to RecQ subfamily helicases that functions as a 3′-5′ DNA helicase in vitro. We now report that the BLM helicase can unwind G4 DNA. The BLM G4 DNA unwinding activity is ATP-dependent and requires a short 3′ region of single-stranded DNA. Strikingly, G4 DNA is a preferred substrate of the BLM helicase, as measured both by efficiency of unwinding and by competition. These results suggest that G4 DNA may be a natural substrate of BLM in vivo and that the failure to unwind G4 DNA may cause the genomic instability and increased frequency of sister chromatid exchange characteristic of Bloom’s syndrome.

Dynamic roles for G4 DNA in the biology of eukaryotic cells

Recent advances have made a persuasive case for the existence of G4 DNA in living cells, but what—if any—are its functions? Experiments have established how G4 DNA may contribute to the biology of eukaryotic cells, and genomic analysis has identified new ways in which the potential to form G4 DNA may influence gene regulation and genomic stability. This Perspective highlights those advances and identifies some key open questions.

Gene function correlates with potential for G4 DNA formation in the human genome.

G-rich genomic regions can form G4 DNA upon transcription or replication. We have quantified the potential for G4 DNA formation (G4P) of the 16 654 genes in the human RefSeq database, and then correlated gene function with G4P. We have found that very low and very high G4P correlates with specific functional classes of genes. Notably, tumor suppressor genes have very low G4P and proto-oncogenes have very high G4P. G4P of these genes is evenly distributed between exons and introns, and it does not reflect enrichment for CpG islands or local chromosomal environment. These results show that genomic structure undergoes selection based on gene function. Selection based on G4P could promote genomic stability (or instability) of specific classes of genes; or reflect mechanisms for global regulation of gene expression.

The G4 Genome

Recent experiments provide fascinating examples of how G4 DNA and G4 RNA structures—aka quadruplexes—may contribute to normal biology and to genomic pathologies. Quadruplexes are transient and therefore difficult to identify directly in living cells, which initially caused skepticism regarding not only their biological relevance but even their existence. There is now compelling evidence for functions of some G4 motifs and the corresponding quadruplexes in essential processes, including initiation of DNA replication, telomere maintenance, regulated recombination in immune evasion and the immune response, control of gene expression, and genetic and epigenetic instability. Recognition and resolution of quadruplex structures is therefore an essential component of genome biology. We propose that G4 motifs and structures that participate in key processes compose the G4 genome, analogous to the transcriptome, proteome, or metabolome. This is a new view of the genome, which sees DNA as not only a simple alphabet but also a more complex geography. The challenge for the future is to systematically identify the G4 motifs that form quadruplexes in living cells and the features that confer on specific G4 motifs the ability to function as structural elements.

Conserved elements with potential to form polymorphic G-quadruplex structures in the first intron of human genes

To understand how potential for G-quadruplex formation might influence regulation of gene expression, we examined the 2 kb spanning the transcription start sites (TSS) of the 18 217 human RefSeq genes, distinguishing contributions of template and nontemplate strands. Regions both upstream and downstream of the TSS are G-rich, but the downstream region displays a clear bias toward G-richness on the nontemplate strand. Upstream of the TSS, much of the G-richness and potential for G-quadruplex formation derives from the presence of well-defined canonical regulatory motifs in duplex DNA, including CpG dinucleotides which are sites of regulatory methylation, and motifs recognized by the transcription factor SP1. This challenges the notion that quadruplex formation upstream of the TSS contributes to regulation of gene expression. Downstream of the TSS, G-richness is concentrated in the first intron, and on the nontemplate strand, where polymorphic sequence elements with potential to form G-quadruplex structures and which cannot be accounted for by known regulatory motifs are found in almost 3000 (16%) of the human RefSeq genes, and are conserved through frogs. These elements could in principle be recognized either as DNA or as RNA, providing structural targets for regulation at the level of transcription or RNA processing.

High Affinity Interactions of Nucleolin with G-G-paired rDNA

Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is K D = 1 nm. Two separate domains of nucleolin can interact with G-G-paired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-G-paired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination.

G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, a role for G-G pairing in immunoglobulin switch recombination

The immunoglobulin heavy chain switch regions contain multiple runs of guanines on the top (nontemplate) DNA strand. Here we show that LR1, a B cell-specific, duplex DNA binding factor, binds tightly and specifically to synthetic oligonucleotides containing G-G base pairs (K D ≤ 0.25 nm). LR1 also binds to single-stranded G-rich sequences (K D ≈ 10 nm). The two subunits of LR1, nucleolin and hnRNP D, bind with high affinity to G4 DNA (K D = 0.4 and 0.5 nm, respectively). LR1 therefore contains two independent G4 DNA binding domains. We propose that LR1 binds with G-G-paired structures that form during the transcription of the S regions that is prerequisite to recombination in vivo. Interactions of donor and acceptor S regions with subunits of the LR1 could then juxtapose the switch regions for recombination.

Somatic hypermutation: How many mechanisms diversify V region sequences?

lmmunoglobulin variable (V) region sequences are tailored to recognize antigen by the process of somatic hypermutation. Somatic hypermutation of V regions can occur either before or after challenge with antigen. When somatic hypermutation occurs before challenge with antigen, the result is to increase the diversity of the preimmune repertoire. When somatic hypermutation occurs in response to antigen stimulation, it is coupled with selection for antigen binding, and the result is to increase antibody affinity for a specific antigen. Insights into mechanism can sometimes come from studying related processes in a variety of organsims. Here I review some similarities between antigen-independent and antigen-driven somatic hypermutation and suggest that variations upon a single molecular mechanism might produce the distinct patterns of templated and untemplated mutation that characterize somatic hypermutation in different organisms. Antigen-Driven Somatic Hypermutation: Mutation Coupled with Selection Somatic hypermutation has been most intensively studied in mice, where hypermutation occurs after challenge with antigen and targets single base changes to the rearranged V regions. The rate of hypermutation approaches 10m3 per base pair per generation, some 105-fold higher than the mutation rate for an untargeted locus in the same cell. Hypermutation is coupled with selection for antigen binding, and a loto lOO-fold increase in affinity for specific antigen distinguishes the very good antibodies of the primary response (Kd, 1 Om7 M) from the extraordinary antibodies of the secondary response (Kd, lo-@ to 10m9 M). This dramatic increase in affinity can result from as few as two or three amino acid substitutions in a 100 residue V domain. Antigen-driven somatic hypermutation occurs in highly organized microenvironments, called germinal centers, where hypermutation is coupled with selection for antigen binding. Germinal centers develop in the follicles of the peripheral lymphoid organs following challenge with antigen (reviewed by Nossal, 1991; MacLennan, 1994). Visualized in sections of lymph node or spleen, germinal centers consist of a mantle surrounding a histologically distinct dark zone and a light zone. Small, resting B cells compose the mantle, and these cells express a large and diverse repertoire of unmutated V region sequences. B cells selected for antigen recognition populate the dark zone, where they proliferate and where somatic hypermutation occurs. Descendants of dark zone B cells migrate to the light zone, cease proliferating, and reveal their newly altered surface immunoglobulin molecules. Antigen displayed on the web of follicular dendritic cells in the light zone can then mediate affinity selection. Distinct surface markers characterize germinal center B cells at different stages of development, and this has recently permitted fractionation of germinal center B cells into distinct populations (Pascual et al., 1994) a critical step in studying both the biology and biochemistry of hypermutation. In addition to B cells, a few T cells also reside within the germinal centers. These T cells were long thought to regulate B cell hypermutation while being immune to the hypermutation process themselves. Recently, however, Kelsoe and collaborators reported that Va (but not V(3) regions of the T cell receptor genes undergo hypermutation in germinal centers (Zheng et al., 1994). T ceil receptor hypermutation is surprising and, not surprisingly, controversial (see Bach1 et al., 1995; Kelsoe et al., 1995). Although immunoglobulin gene hypermutation can be readily rationalized, T cell hypermutation seems dangerous-if these cells return to the periphery, they may exhibit new and possibly autoreactive specificities. Hotspots for Hypermutation In hypermutated murine V region sequences, mutations are not evenly distributed throughout the V region but are concentrated in the complementarity determining regions (CDRs), which encode the amino acids that make contact with antigen (see Figure 1). Clustering of hypermutation in the CDRs was noticed when the first V regions were sequenced. The ready explanation was that affinity selection must enrich for B cells carrying mutations in the regions that encode the antigen-binding site. However, it has recently been shown that targeting of somatic hypermutation to the CDRs is a property of the hypermutation mechanism itself (Betz et al., 1993a, 1993b; GonzalezFernandez and Milstein, 1993; Gonzalez-Fernandez et al., 1994; Yelamos et al., 1995). To separate the intrinsic properties of the hypermutation process from the effects of selection for antigen binding, Milstein, Neuberger, and their colleagues began by amassing a sequence database of VKOX~ light chain V regions that had undergone somatic hypermutation without affinity selection. These VK regions had escaped selection because they were carried as “passenger” trans genes that did not contribute to an antigen-specific immune response (Betz et al., 1993a, 1993b; GonzalezFernandez and Milstein, 1993). Sequences of many such hypermutated but unselected genes revealed a very strong intrinsic hotspot in CDRl of the VKOX~ region. Sub-

AID binds to transcription-induced structures in c- MYC that map to regions associated with translocation and hypermutation

Translocation and aberrant hypermutation of c-MYC are common in B-cell lymphomas. Activation-induced Cytidine Deaminase (AID) initiates switch recombination and somatic hypermutation in B cells by targeted deamination of transcribed genes. We show that transcription of the immunoglobulin S regions and c-MYC results in formation of similar DNA structures, ‘G-loops’, which contain a cotranscriptional RNA: DNA hybrid on the C-rich strand and single-stranded regions and G4 DNA on the G-rich strand. AID binds specifically to G-loops within transcribed S regions and c-MYC, and G-loops in c-MYC map to the regions associated with translocation breakpoints and aberrant hypermutation in B-cell lymphomas. Aberrant targeting of AID to DNA structures formed upon c-MYC transcription may therefore contribute to the genetic instability of c-MYC in B-cell malignancies.

Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease

Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at ≈1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.