AID in antibody perfection.

Expressed immunoglobulin (Ig) genes undergo alterations in sequence and genomic structure in order to optimize antibody function. A single B cell-specific factor, activation-induced deaminase (AID), initiates these changes by deamination of cytosine to uracil. Uracil in DNA is encountered commonly, and conserved pathways are responsible for its faithful repair. However, at the Ig loci of B cells, AID-initiated damage is processed to produce three distinct outcomes: somatic hypermutation, class switch recombination and gene conversion. This review focuses on the role of AID in Ig gene diversification, emphasizing how AID functions within the mechanism of the Ig gene diversification pathway; and highlights open questions for future research, particularly the most provocative current question: what makes a gene a target for AID-initiated mutagenesis?

A human nuclease specific for G4 DNA.

We have identified a human nuclease that specifically cleaves four-stranded DNA stabilized by G quartets (G4 DNA). This nuclease, GQN1 (G quartet nuclease 1), cuts within the single-stranded region 5' of the barrel formed by stacked G quartets. GQN1 does not cleave duplex or single-stranded DNA, Holliday junctions, or G4 RNA. Cleavage depends on DNA structure and not on flanking sequence. Activity is elevated in but not restricted to B cells, making GQN1 a strong candidate for function in immunoglobulin heavy chain class switch recombination. Identification of a mammalian nuclease that specifically cleaves G4 DNA provides further support for the notion that DNA structures stabilized by G quartets form in vivo and function in regulated recombination and genomic evolution.

DNA breaks in hypermutating immunoglobulin genes: evidence for a break-and-repair pathway of somatic hypermutation.

To test the hypothesis that immunoglobulin gene hypermutation in vivo employs a pathway in which DNA breaks are introduced and subsequently repaired to produce mutations, we have used a PCR-based assay to detect and identify single-strand DNA breaks in lambda1 genes of actively hypermutating primary murine germinal center B cells. We find that there is a two- to threefold excess of breaks in lambda1 genes of hypermutating B cells, relative to nonhypermutating B cells, and that 1.3% of germinal center B cells contain breaks in the lambda1 gene that are associated with hypermutation. Breaks were found in both top and bottom DNA strands and were localized to the region of lambda1 that actively hypermutates, but duplex breaks accounted for only a subset of breaks identified. Almost half of the breaks in hypermutating B cells occurred at hotspots, sites at which two or more independent breaks were identified. Breaksite hotspots were associated with characteristic sequence motifs: a pyrimidine-rich motif, either RCTYT or CCYC; and RGYW, a sequence motif associated with hypermutation hotspots. The sequence motifs identified at breaksite hotspots should inform the design of substrates for characterization of activities that participate in the hypermutation pathway.

Substrate-specific inhibition of RecQ helicase.

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K(i)) for NMM inhibition of G4 DNA unwinding is 1.7 microM, approximately two orders of magnitude below the K(i) for inhibition of duplex DNA unwinding (>100 microM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells.

DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers to increase the specificity and sensitivity of amplification. Breaksites are then mapped by batch sequencing LM-PCR products. This allows easy identification of multiple breaksites per reaction without tedious fractionation of PCR products by gel electrophoresis or cloning. Breaksite batch mapping requires little starting material and can be used to identify either single- or double-strand breaks.

The C-terminal domain of nucleolin accelerates nucleic acid annealing.

We report that the abundant nucleolar protein nucleolin accelerates nucleic acid annealing. Nucleolin accelerates annealing of complementary oligonucleotides and of oligonucleotides that contain a limited number of mismatches. The annealing activity of nucleolin can be localized to a C-terminal region consisting of two RNA binding domains (RBD3 and RBD4) and the RGG(9) domain (RBD3-RBD4-RGG(9)). This same region mediates self-association of nucleolin. The RGG(9) domain of nucleolin, believed to mediate interactions between nucleolin and several ribosomal proteins, is neither sufficient for self-association, as determined by small-angle X-ray scattering, nor can it independently accelerate annealing. Acceleration of nucleic acid annealing by nucleolin is likely to depend on self-association of nucleolin molecules bound to nucleic acid.

Transcriptional activation by LR1 at the Emu enhancer and switch region sites.

LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Emu immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Emu core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Emu core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions.

In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance.

Mammalian chromosomes terminate with a 3' tail which consists of reiterations of the G-rich repeat, d(TTAGGG). The telomeric tail is the primer for replication by telomerase, and it may also invade telomeric duplex DNA to form terminal lariat structures, or T loops. Here we show that the ubiquitous and highly conserved mammalian protein hnRNP D interacts specifically with the G-rich strand of the telomeric repeat. A single gene encodes multiple isoforms of hnRNP D. All isoforms bind comparably to the G-rich strand, and certain isoforms can also bind tightly and specifically to the C-rich telomeric strand. G-rich telomeric sequences readily form structures stabilized by G-G pairing, which can interfere with telomere replication by telomerase. We show that hnRNP D binding to the G-rich strand destabilizes intrastrand G-G pairing and that hnRNP D interacts specifically with telomerase in human cell extracts. This biochemical analysis suggest that hnRNP D could function in vivo to destabilize structures formed by telomeric G-rich tails and facilitate their extension by telomerase.

HuR binding to cytoplasmic mRNA is perturbed by heat shock.

AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)(+) RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)(+) RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.

Secret sharers in the immune system: a novel RNA editing activity links switch recombination and somatic hypermutation.

A new mechanism for regulation in the immune system has been identified: a cytidine deaminase is critical for both class switch recombination and somatic hypermutation, revealing an unanticipated link between these two processes.

Coordinated response of mammalian Rad51 and Rad52 to DNA damage.

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)-Rad52, we now show that DNA damage causes Rad51 and GFP-Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP-Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.

Activation and targeting of immunoglobulin switch recombination by activities induced by EBV infection.

EBV is strongly associated with Burkitt's lymphoma, a B cell malignancy. In certain types of Burkitt's lymphoma, the c-myc gene has undergone translocation to the S regions associated with heavy chain switch recombination. It has not been established whether EBV infection induces recombination activities, which in turn promote translocation of c-myc, or whether translocation precedes viral infection and provides a growth advantage that is further enhanced by factors encoded or induced by the virus. To distinguish between these possibilities, we have compared the level of switch recombination activities in the EBV-negative lymphoma, BJAB, and in its EBV-infected derivative, BJAB-B1, in experiments that assayed recombination of an extrachromosomal switch substrate during transient transfection. We have found that BJAB-B1 and other EBV-positive B cell lines supported high levels of recombination of switch substrates, to produce junctions like those found in products of chromosomal switch recombination. In contrast, BJAB did not support comparable levels of switch substrate recombination. In EBV-positive B cell lines, the ability to support switch substrate recombination correlated with levels of LR1, a B cell-specific factor which is a transcriptional regulator of c-myc and which also appears to function in switch recombination. Our observations support the hypothesis that EBV infection can induce activities that affect switch recombination and thus contribute to the translocations of c-myc to the S regions that characterize certain classes of lymphomas.

Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage.

The importance of RAD52 in establishment and maintenance of genomic structure has been established by genetic experiments in the yeast Saccharomyces cerevisiae, where mutation of RAD52 has been shown to diminish DNA repair and recombination of a variety of markers, including the rDNA [1] [2] [3]. Biochemical analysis has shown that yeast and mammalian Rad52 proteins have some identical functions in vitro [4] [5] [6], but targeted deletion of Rad52 in vertebrates has little effect on repair and recombination [7] [8]. These results raise the question of whether mammalian Rad52 does indeed function in recombination and/or repair. Here we show that Rad52 is distributed throughout the nucleoplasm in actively cycling mammalian cells and is localized specifically to the nucleoli in S phase. In response to ionizing radiation, Rad52 relocalizes to form distinctive foci which are distributed throughout the nucleus and which colocalize with Rad50 foci in the DNA damage response. These data suggest that rDNA recombination and DNA repair are functions shared by mammalian Rad52 and its S. cerevisiae homolog, and provide evidence for the coordinated action of Rad50 and Rad52 in DNA repair.

PMS2-deficiency diminishes hypermutation of a lambda1 transgene in young but not older mice.

The Pms2 gene is involved in DNA mismatch repair in mammalian cells, and has recently been shown to affect hypermutation of mammalian immunoglobulin genes. We have studied hypermutation of a lambda1 transgene in chronically stimulated Peyer's patch B cells of both young and old mice deficient in function of Pms2. In young (3-4 months) mice, somatic hypermutation is fourfold lower in PMS2-deficient mice than in control mice. This difference is statistically significant (P < 0.05). In contrast, in older mice (9 months of age), hypermutation levels are indistinguishable in the Pms2-/- and Pms2+/+ backgrounds. In the older mice, there was no clear difference in the fraction of clones carrying either any mutations or at least two mutations when PMS2-deficient mice were compared with their wild-type littermates. As genomic instability increases with age, this observation is difficult to reconcile with the hypothesis that highly mutated B cells cannot survive in Peyer's patches. Moreover, there were clear differences apparent in the mutation spectra of the Pms2-/- and Pms2+/+ mice. In the PMS2-deficient background, deletion and insertion mutations were found, and there was a significant decrease in the ratio of A mutations to T mutations in comparison with the Pms2+/+ controls. Our data support the hypothesis that PMS2 functions in somatic hypermutation, and are most consistent with the hypothesis that the role of PMS2 is direct rather than indirect.

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 KD = 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.

The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs.

The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase.