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M R Lieber

Publications and source records attributed to M R Lieber.

At least 19 recordsLinked to original sources
PubMed1996

Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.

Antibodies, Monoclonal↗
PubMed1996

Asymmetric mutation around the recombination break point of immunoglobulin class switch sequences on extrachromosomal substrates.

Junctions at class switch recombination sites in the genome are characterized by a unique sequence feature. Nucleotide substitutions and small deletions are common on either of the two sides of the switch junction, but not on both together. We have previously reported an extrachromosomal substrate assay system for analyzing the recombination of class switch sequences. Here we have sequenced nine junctions on each side of the break point and compared these to 17 recombination junctions of control substrates from the same cells. Five of the nine switch recombination junctions have nucleotide substitutions and deletions, with multiple nucleotide changes being more common. Furthermore, mutations were found only on a single side of the junction, just as for the recombination of switch sequences in the genome. In contrast, only one of 17 control substrate junctions had a mutation, and this was a single nucleotide insertion. This difference is highly significant (P < 0.00007) and indicates that the fundamental recombination mechanism is likely to be similar for switch sequences in the chromosome and on minichromosome substrates.

Base Sequence↗
PubMed1996

Mechanistic constraints on diversity in human V(D)J recombination.

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.

Animals↗
PubMed1995

RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination.

Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination. We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions. In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments. We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels. These altered mobilities are resistant to RNase A but sensitive to RNase H. Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA. Importantly, complex formation only occurs upon transcription in the physiologic orientation. Reaban and Griffin [1990 Nature, 348, 342-344] found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment. Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand. These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.

Animals↗
PubMed1995

Human DNA-activated protein kinase (DNA-PK) is homologous to phosphatidylinositol kinases.

DNA-activated protein kinase (DNA-PK) is a serine/threonine protein kinase that interacts with a DNA end-binding heterodimeric protein, Ku, and is activated by double-stranded DNA. Genomic clones that contain the DNA-PK gene complement the murine scid defect, indicating that DNA-PK affects double-strand break repair and V(D)J recombination. Here we describe the cDNA sequence of the region that corresponds to about 100 kDa of C-terminal sequence of this large (> p350 kDa) protein. This region contains a kinase domain that has strong homology to phosphatidylinositol kinases.

1-Phosphatidylinositol 4-Kinase↗
PubMed1995

Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen.

The 5'-->3'-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5'-->3'-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.

Base Sequence↗
PubMed1995

Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences.

B-lymphocyte-specific class switch recombination is known to occur between pairs of 2- to 10-kb switch regions located immediately upstream of the immunoglobulin constant heavy-chain genes. Others have shown that the recombination is temporally correlated with the induction of transcription at the targeted switch regions. To determine whether this temporal correlation is due to a mechanistic linkage, we have developed an extrachromosomal recombination assay that closely recapitulates DNA deletional class switch recombination. In this assay, the rate of recombination is measured between 24 and 48 hr posttransfection. We find that recombinants are generated in a switch sequence-dependent manner. Recombination occurs with a predominance within B-cell lines representative of the mature B-cell stage and within a subset of pre-B-cell lines. Transcription stimulates the switch sequence-dependent recombination. Importantly, transcription activates recombination only when directed in the physiologic orientation but has no effect when directed in the nonphysiologic orientation.

Animals↗
PubMed1995

The scid factor on human chromosome 8 restores V(D)J recombination in addition to double-strand break repair.

The murine severe combined immune deficiency mutation (scid) is characterized by a lack of B- and T-lymphoid cells due to a defect in lymphoid V(D)J recombination. Moreover, defective rejoining of DNA double-strand breaks (dsb) in scid cells also results in a marked increase in sensitivity to ionizing radiation. Recently, the putative human homologue of the murine scid gene locus, HYRC1, was assigned to human chromosome 8q11, based on the radiation sensitivity of scid cells as compared to scid:human cell hybrids carrying portions of human chromosome 8. Given the precedent (e.g., ataxia-telangiectasia) for genes other than the affected one being able to complement radiation defects, we were interested in determining if the V(D)J recombination defect was also corrected by the HYRC1 locus. The V(D)J recombination analysis using extrachromosomal DNA substrates in control scid cells (SC3VA2) versus complemented cells (RD13B2) indicates that the radiation sensitivity-complemented cells (RD13B2) are also fully complemented for the V(D)J recombination reaction, whereas the control (uncomplemented) cells (SC3VA2) fail to carry out V(D)J recombination normally. Slightly over 60% of the radiation-induced dsb are rejoined even in scid cells, and this alternative pathway is temperature sensitive. Only the remaining 30-35% of dsb require the introduction of the HYRC1 locus, and this pathway is not temperature sensitive. This merely partial contribution of the scid factor to the repair process suggests the presence of another pathway of dsb repair. Our results indicate that the HYRC1 locus, assigned to human chromosome 8q11, encodes the scid factor, which is involved in all V(D)J recombination coding joint formation and in 30-35% of dsb repair by the temperature-resistant pathway.

Animals↗
PubMed1995

DNA structural elements required for FEN-1 binding.

In eukaryotic cells, a 5'-flap DNA endonuclease and a double-stranded DNA 5'-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)'-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the Fadj strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the Fadj strand using double flap structures reveals that the Fadj strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.

Base Sequence↗
PubMed1995

Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human.

We recently purified and cloned the gene for a DNA structure-specific endonuclease, FEN-1, from murine cells. The murine protein recognizes 5' DNA flap structures that have been proposed in DNA replication, repair, and recombination. Here, we report the sequence of the human FEN1 gene. The translated sequence is identical to peptide sequence obtained from maturation factor-1, which is 1 of the 10 essential proteins for cell-free DNA replication. The human protein has the same structure-specific DNA endonuclease activity as the murine protein. Two human chromosomal hybridization signals, 11q12 and 1p22.2, were observed by FISH analysis using human genomic clones homologous to the mouse Fen-1 gene. The localization on human 11q12 was confirmed using radiation-reduced hybrids. The mouse Fen-1 gene is assigned to chromosome 19 based on somatic cell hybrids. The significance of these FEN1 gene localizations in human and mouse is discussed.

Amino Acid Sequence↗
PubMed1994

Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA.

Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

Animals↗
PubMed1994

Distinct roles for RAG-1 in the initiation of V(D)J recombination and in the resolution of coding ends.

Although RAG-1 and RAG-2 have been shown to be indispensible for V(D)J recombination, their exact role in this reaction remains unclear. Co-transfecting RAG-1 and RAG-2 expression vectors into NIH3T3 fibroblasts confers V(D)J recombination activity to these otherwise recombinationally inactive cells. In this report we have found that in transient transfections of mouse NIH3T3 fibroblasts with RAG-1 and RAG-2 and the appropriate recombination substrates, one RAG-1 expression vector, pRAG-1A, is capable of yielding both signal joints and coding joints, while another RAG-1 expression vector, pRAG-1B, yields only signal joints. The RAG-1 open reading frame for these two expression vectors is interchangeable, indicating that the inability to resolve coding joints is due to the 45-base pair difference found in the 5'-untranslated regions of these constructs. Differences in this region result in a 15-fold difference in gene expression when the luciferase coding region is substituted for the RAG-1 cDNA. This report provides evidence that RAG-1 may have a role in both the initiation of V(D)J recombination as well as the resolution of coding ends. The data also suggest that these RAG-1 activities may be dependent on different levels of RAG-1 expression.

3T3 Cells↗
PubMed1994

Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.

Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair. Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures. Here, we describe the cloning of the murine FEN-1 gene. The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2. We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases. The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair. The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.

Amino Acid Sequence↗
PubMed1994

Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes.

We used the polymerase chain reaction (PCR) to amplify genes encoding murine immunoglobulin (Ig) lambda light-chain variable (V) regions, using DNA isolated from populations of germinal center B-cells, to study somatic hypermutation at this locus. Sequence analysis revealed that 30% of the amplified products were chimeric molecules consisting of segments of the V lambda 1 and V lambda 2 genes. Furthermore, an amplification- and cloning-associated artifact exchanged sequences between mutational variants of V lambda 1 genes. These PCR artifacts interfere with the analysis of somatic hypermutation of Ig genes. An alternative method that avoids these artifacts is suggested which involves the amplification of individual V lambda genes from single cells.

Animals↗
PubMed1994

Transcription, topoisomerases and recombination.

Transcription, DNA topoisomerases and genetic recombination are interrelated for several structural reasons. Transcription can affect DNA topology, resulting in effects on recombination. It can also affect the chromatin structure in which the DNA resides. Topoisomerases can affect DNA and/or chromatin structure influencing the recombination potential at a given site. Here we briefly review the extent to which homologous direct repeat recombination and site-specific recombination in eukaryotes are affected by transcription and topoisomerases. In some cases, transcription or the absence of topoisomerases have little or no effect on recombination. In others, they are important components in the recombinational process. The common denominator of any effects of transcription and topoisomerases on recombination is their shared role in altering DNA topology.

Animals↗
PubMed1994

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.

Animals↗
PubMed1994

Analysis of individual immunoglobulin lambda light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation.

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenvironment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse lambda light chain genes which arose in response to a specific antigen. Laboratory mice possess three V lambda subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single lambda light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V lambda 1 genes from these single cells for mutational analysis. None of the 32 V lambda 1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other V lambda subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.

Amino Acid Sequence↗