PubMed Health⌕ Search

Biomedical subjects

D Scherly

Publications and source records attributed to D Scherly.

17 recordsLinked to original sources
PubMed1994

Isolation of active recombinant XPG protein, a human DNA repair endonuclease.

Complementation group G of xeroderma pigmentosum (XP-G) is one of the most rare and phenotypically heterogeneous forms of this inherited disorder. XP-G patients vary from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological complications of Cockayne's syndrome. The XPG gene encodes an acidic protein with a predicted molecular mass of 133 kDa that confers normal UV resistance when expressed in XP-G cells. Here we report the isolation of full-length XPG as a soluble protein expressed from a recombinant baculovirus. The purified polypeptide corrects the DNA nucleotide excision repair defect of XP-G cell extracts in vitro, and it acts as a magnesium-dependent single-stranded DNA endonuclease. This is the first direct evidence for a human protein with properties that implicate it in the incision step of nucleotide excision repair.

Animals↗
PubMed1993

La proteins from Xenopus laevis. cDNA cloning and developmental expression.

In mammalian nuclei, newly-synthesized RNA polymerase III transcripts are transiently associated with a phosphorylated polypeptide of approximately 50 kDa called the La protein. Here we provide evidence that the frog Xenopus laevis contains mRNAs for two highly related La proteins, each apparently encoded by a single gene. Both forms of the La protein contain the RNP-80 motif previously identified in many RNA binding proteins. The steady state levels of La mRNAs and protein are approximately constant in oocytes, eggs and embryos. This implies a progressive and severe decrease in these levels on a per cell basis during early development. In particular, neither the La mRNA nor protein level increases at the mid-blastula transition, the time when RNA polymerase III transcription first occurs during embryogenesis.

Amino Acid Sequence↗
PubMed1993

Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

Amino Acid Sequence↗
PubMed1991

Analysis of in vitro binding of U1-A protein mutants to U1 snRNA.

Despite the great sequence similarity between U1A and U2B", both proteins do have a difference in RNA binding specificity and in the way they bind to their cognate RNAs. The U1A protein is able to bind in vitro U1 RNA independently of other factors. The U2B" protein binds specifically to U2 RNA in the presence of the U2A' protein only. We have compared the effect on RNA binding of multiple double point mutations at analogous positions in the U1A and U2B" protein. The results obtained show that amino acids at almost all of the analogous positions tested in U1A and U2B" have a comparable qualitative effect on RNA binding although the quantitative effect of mutations on U2B" is more severe than on U1A. Using U1A mutants with internal duplications a distinct area of the RNP motif of the U1A protein was identified which appears not to be directly involved in U1 RNA binding. In addition, roles of the highly conserved RNP1 and RNP2 sequences of the N-terminal RNP motif of the U1A protein, are investigated by replacing them with the analogous U1-70K sequences.

Binding Sites↗
PubMed1991

Conserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding.

By the use of hybrids between a U1 small nuclear ribonucleoprotein (snRNP: U1A) and a U2 snRNP (U2B") we have identified regions containing 29 U1A-specific amino acid residues scattered throughout the 117 N-terminal residues of the protein, which are involved in binding to U1 RNA. The U1A-specific amino acid residues have been arbitrarily divided into seven contiguous groups. None of these groups is sufficient for U1 binding when transferred singly into the U2B" context, and none of the groups is essential for U1 binding in U1A. Several different combinations of two or more groups can, however, confer the ability to bind U1 RNA to U2B", suggesting that most or all of the U1A-specific amino acid residues contribute incrementally to the strength of the specific binding interaction. Further evidence for the importance of the U1A-specific amino acid residues, some of which lie outside the region previously shown to be sufficient for U1 RNA binding, is obtained by comparison of the sequence of human and Xenopus laevis U1A cDNAs. These are extremely similar (94.4% identical) between amino acid residues 7 and 114 but much less conserved immediately upstream and downstream from this region.

Amino Acid Sequence↗
PubMed1991

A weak interaction between the U2A' protein and U2 snRNA helps to stabilize their complex with the U2B" protein.

The U2 snRNP complex contains two specific proteins, U2B" and U2A'. We have analysed the interaction of U2A' with U2B" and with U2 RNA. U2A' can form an weak but detectable RNA-protein complex with U2 RNA and a stable protein complex with U2B". This protein-protein complex binds efficiently and specifically to U2 RNA. Binding experiments with mutant forms of U2A' shows that the region of U2A' essential for binding to U2B" is extensive, being located between amino acid position 1-164. The behaviour of the wild type U2A' protein, and in particular of a mutant version of the protein in which amino acids 3, 4 and 5 are mutated, suggests that U2A' forms a weak interaction with U2 RNA which helps to stabilize the U2A'-U2B"-U2 RNA complex. Mutants of U2 RNA were used to localize the region of U2 RNA important for interaction with U2A'. The results show that U2A' interacts with the stem of hairpin IV.

Cloning, Molecular↗
PubMed1990

Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs.

The basis of the specificity of interaction of U1 and U2 small nuclear (sn)RNAs and their cognate binding proteins, U1A and U2B'', has been examined. The U1A protein recognizes U1 snRNA on its own, whereas U2B'' binds specifically to U2 snRNA only in the presence of a second protein, U2A'. Exchange of two nucleotides between the two RNAs or of eight amino acids between the two proteins reverses binding specificity.

Amino Acid Sequence↗
PubMed1990

The U2B'' RNP motif as a site of protein-protein interaction.

The U2 snRNP contains two specific proteins, U2B'' and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B'' and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B'' shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1-88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B'' fragment is capable of making specific protein--RNA and protein-protein interactions its structure, as measured by the ability to bind to U2A'', appears to depend on the rest of the protein. Hybrids between U2B'' and the closely related U1A protein are used to localize U2B'' specific amino acids involved in protein-protein interaction. These can be divided into two functional groups. U2A' interaction with U2B'' amino acids 37-46 permits binding to U2 RNA whereas interaction with U2B'' specific amino acids between positions 14 and 25 reduces non-specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein-protein interaction in the assembly of RNPs, particularly since the region of U2'' involved in interaction with U2A' consists mainly of a conserved RNP motif.

Amino Acid Sequence↗
PubMed1990

Multiple domains of U1 snRNA, including U1 specific protein binding sites, are required for splicing.

Domains of U1 snRNA which are functionally important have been identified using a splicing complementation assay in Xenopus oocytes. Mutations in, and deletions of, all three of the hairpin loop structures near the 5' end of the RNA are strongly deleterious. Similarly, mutation of the Sm binding site abolishes complementation activity. Analysis of the protein binding properties of the mutant U1 snRNAs reveals that three of the functionally important domains, the first two hairpin loops and the Sm binding site, are required for interaction with U1 snRNP proteins. The fourth functionally important domain does not detectably affect snRNP protein binding and is not evolutionarily conserved. All of the deleterious mutations are shown to have similar effects on in vivo splicing complex formation.

Animals↗
PubMed1989

Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA.

The interaction between the U1 snRNP-specific U1 A protein and U1 snRNA has been analysed. The binding site for the protein on the RNA is shown to be in hairpin II, which extends from positions 48 to 91 in the RNA. Within this hairpin the evolutionarily conserved loop sequence is crucial for interaction with U1 A protein. U1 A protein can also bind the loop sequence when it is part of an artificial RNA which cannot form a stable hairpin structure. The region of the protein required to bind to U1 snRNA consists of a conserved 80 amino acid motif, previously identified in many ribonucleoprotein (RNP) proteins, together with (maximally) 11 N-terminal and 10 C-terminal flanking amino acids. Point mutations introduced into two of the most highly conserved regions of this motif abolish RNA binding. U1 snRNA mutants from which the U1 A binding site has been deleted are shown to be capable of assembly into RNP particles which are immunoprecipitable by patient antisera which recognize U1 A protein. The role of RNA-protein and protein-protein interactions in U snRNP assembly are discussed.

Animals↗
PubMed1988

Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K).

A cDNA and two genes for the Xenopus laevis U snRNP 70K protein have been cloned and partially sequenced. The cDNA encodes a protein whose predicted mol. wt is 57 kd but which migrates as a 70 kd protein in SDS-PAGE when translated in vitro from a cDNA transcript. The predicted protein sequences of the human and Xenopus U1 70K are shown to be very similar. Analysis of several genomic clones suggests that there are at least two, and possibly more, different genes coding for the 70K protein in the Xenopus genome. The two genes analysed in detail cover approximately 16 kb and are divided into 10 exons of which the last exon covers more than half of the protein coding sequence. During Xenopus development several different stage-specific RNAs hybridizing to the U1 70K cDNA are detected. The promoter region of one of the cloned genes is demonstrated to be functionally active, and to show apparent differences from other pol II promoters.

Amino Acid Sequence↗
PubMed1987

Structure and transcription termination of a lysine tRNA gene from Xenopus laevis.

Termination of RNA polymerase III transcripts commonly occurs at clusters of T residues. A T4 tract located 72 base-pairs beyond a lysine tRNA gene from Xenopus laevis serves as an efficient termination site for the tRNA(Lys) precursors synthesized from this gene in homologous cell-free extracts. Nucleotides following this T tract influence the extent of read-through transcription in vitro, but in a way that differs from Xenopus 5 S RNA termination. Only approximately 50% of the transcripts initiated in vitro extend as far as this downstream T cluster. The remainder prematurely terminate at a second T4 tract located within the gene itself. The contrasting behaviour of these two T tracts in injected oocytes indicates that termination can be influenced by more than just RNA polymerase III alone, and that different components may contribute to, or hinder, termination at these sites. Prematurely terminated tRNA(Lys) transcripts are detectable in RNA from ovary tissue but not from a kidney cell line, suggesting that read-through transcription beyond intragenic T clusters can be modulated in vivo.

Animals↗