Erik Selsing

Structures for the polynucleotide complexes poly(dA) · poly(dT) and poly(dT) · poly(dA) · poly(dT)

X-ray diffraction analyses of fibers of polydeoxyadenylic acid · polydeoxythymidylic acid show that this molecule exists as a 10-fold double-helix with axial rise per nucleotide h = 3.24 to 3.29 A. The structure is very similar to B-DNA (h = 3.37 A) in having C3-exo furanose rings and base-pairs positioned centrally on the helix axis, but distinctive enough to have two packing modes, neither of which has been observed for B-DNA. Although the triple-stranded poly(dT) · poly(dA) · poly(dT) also has a large value of h(3.26 A), each of the chains is a 12-fold helix of the A-genus with C3-endo furanose rings and bases displaced several Angstrom units from the helix axis.

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

Abstract A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.

Somatic mutation of immunoglobulin light-chain variable-region genes

A single germline immunoglobulin kappa-variable-region gene, VK167, is rearranged and expressed in two myelomas, MOPC167 and MOPC511. Only this single germline gene displays close homology to the expressed genes. Neither of the rearranged, functional genes, however, has a nucleotide sequence that is identical to the germline VK167 gene. Both active genes display several single-base-pair mutations with respect to the germline sequence. The nucleotide sequence data predict the alteration of a restriction-enzyme-recognition site within the VK167 gene between germline cells and cells producing the MOPC167 light-chain protein. Based on this restriction-site alteration, Southern blot analysis proves unambiguously that no gene present in the germline BALB/c mouse genome contains the exact VK167 nucleotide sequence found in cells committed to MOPC167 antibody production. Instead the alterations found in the expressed MOPC167 and MOPC511 V-region genes have apparently arisen by a process of somatic mutation during cellular differentiation. Since nucleotide alterations are found in framework and hypervariable portions of the variable region, the mechanism of somatic mutation is not limited to hypervariable sequences. In addition, Southern blot hybridization indicates that the observed mutations did not arise by recombinational events, but are single-base-pair substitutions. Based on the distribution of mutations that have been found in expressed immunoglobulin variable-region genes, a model that links the introduction of somatic mutations to DNA replication during the V-J joining event is proposed.

Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon Mismatch Repair

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Sμ mainly in the G1 phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G1 phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SμTR−/− B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.

The conformation of C-DNA

The conformation of C-DNA has been re-examined and shown to be much more like B-DNA than previously supposed.

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

ABSTRACT The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Sμ locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Sμ R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Sμ repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Sμ repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Sμ repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.

The Sμ Tandem Repeat Region Is Critical for Ig Isotype Switching in the Absence of Msh2

Deficiencies of the Msh2 protein or the Smu tandem repeat (SmuTR) sequences each reduce isotype switching in mice by about 2- to 3-fold. We find that switching in mice deficient for both Msh2 and SmuTR is nearly ablated. We propose that the SmuTR provides closely spaced cleavage sites that can undergo switch recombination independent of Msh2, whereas cleavages in sequences flanking the SmuTR require Msh2 processing to allow recombinational joining. We also find that changes in Smu sequences alter the focus of switch junctions within Sgamma sequences, indicating that sequences of switch regions act together in the choice of switch recombination junctions. These findings help to explain the conservation of tandemly repeated switch regions associated with heavy chain constant genes in species capable of switching.

Mismatch Repair Proteins MSH2, MLH1, and EXO1 Are Important for Class-Switch Recombination Events Occurring in B Cells That Lack Nonhomologous End Joining

In the absence of core nonhomologous end-joining (NHEJ) factors, Ab gene class-switch recombination (CSR) uses an alternative end-joining (A-EJ) pathway to recombine switch (S) region DNA breaks. Previous reports showing decreased S-junction microhomologies in MSH2-deficient mice and an exonuclease 1 (EXO1) role in yeast microhomology-mediated end joining suggest that mismatch repair (MMR) proteins might influence A-EJ–mediated CSR. We have directly investigated whether MMR proteins collectively or differentially influence the A-EJ mechanism of CSR by analyzing CSR in mice deficient in both XRCC4 and individual MMR proteins. We find CSR is reduced and that Igh locus chromosome breaks are reduced in the MMR/XRCC4 double-deficient B cells compared with B cells deficient in XRCC4 alone, suggesting MMR proteins function upstream of double-strand break formation to influence CSR efficiency in these cells. Our results show that MLH1, EXO1, and MSH2 are all important for efficient A-EJ–mediated CSR, and we propose that MMR proteins convert DNA nicks and point mutations into dsDNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4− B cells have an increased frequency of direct S junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ–mediated CSR.

Shifts in targeting of class switch recombination sites in mice that lack μ switch region tandem repeats or Msh2

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Sμ tandem repeats show shifts indicating that a 4–5-kb DNA domain (bounded upstream by the Iμ promoter) is accessible for switching independent of Sμ sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4–5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Sμ tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.

Immunoglobulin λ genes

Publisher Summary The lambda (ƛ) gene family found in most laboratory mouse strains is one of the smallest immunoglobulin gene systems. The gene segments that appear to be involved in the synthesis of ƛ light chains in the BALB/c mouse have been isolated, characterized, and sequenced. There are two classes of immunoglobulin light-chain polypeptides, designated as kappa (κ) and ƛ, found among serum antibodies, although individual antibody molecules contain only one of these classes. Both κand ƛ light chains are found in most vertebrates. However, the Κ/ƛ ratio varies widely among different species. In mice and humans, ƛ chains are encoded by gene families located on separate chromosomes. The ƛ genes require variable (V) joining (J), V-J recombination, to produce functional proteins. Instead, the ƛ class of light chains appears to reflect an evolutionary duplication and divergence process that serves to provide additional V-region diversity for the antibody repertoire.