scholarly journals Specific Mismatch Recognition in Heteroduplex Intermediates by p53 Suggests a Role in Fidelity Control of Homologous Recombination

1998 ◽  
Vol 18 (9) ◽  
pp. 5332-5342 ◽  
Author(s):  
Christine Dudenhöffer ◽  
Gabor Rohaly ◽  
Katrin Will ◽  
Wolfgang Deppert ◽  
Lisa Wiesmüller
Keyword(s):  
Homologous Recombination ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Base Pairs ◽  
Recombination Processes ◽  
Quantitative Manner ◽  
Mismatch Recognition ◽  
Dna Substrate ◽  
Recombination Experiments

ABSTRACT We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.

The minimum amount of homology required for homologous recombination in mammalian cells

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

scholarly journals Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 3948-3953 ◽  
Author(s):  
P Bullock ◽  
J Miller ◽  
M Botchan
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Somatic Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Recombination Product ◽  
Homologous Recombination Event ◽  
Sequencing Studies ◽  
Purine Pyrimidine

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.

scholarly journals Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated SV40 T Antigen, Covaries With the Ability to Induce Host DNA Synthesis

Genetics ◽  
1997 ◽  
Vol 146 (4) ◽  
pp. 1417-1428
Author(s):  
Masood A Shammas ◽  
Shujuan J Xia ◽  
Robert J Shmookler Reis
Keyword(s):  
Homologous Recombination ◽  
Dna Synthesis ◽  
Human Fibroblasts ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
Wild Type ◽  
Chromosomal Recombination

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.

scholarly journals The minimum amount of homology required for homologous recombination in mammalian cells.

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258 ◽  
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells

1986 ◽  
Vol 6 (11) ◽  
pp. 3948-3953
Author(s):  
P Bullock ◽  
J Miller ◽  
M Botchan
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Somatic Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Recombination Product ◽  
Homologous Recombination Event ◽  
Sequencing Studies ◽  
Purine Pyrimidine

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.

scholarly journals A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1381-1388 ◽  
Author(s):  
L P Villarreal ◽  
R T White
Keyword(s):  
Simian Virus 40 ◽  
Splice Junction ◽  
Simian Virus ◽  
The Body ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Wild Type ◽  
Normal Position ◽  
Nuclear Rna ◽  
Late Region

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.

Effects of mutation position on frequency of marker rescue by homologous recombination

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader

1984 ◽  
Vol 4 (4) ◽  
pp. 813-816
Author(s):  
A Barkan ◽  
J E Mertz
Keyword(s):  
Nucleotide Sequence ◽  
Direct Evidence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
The Body ◽  
Size Distributions ◽  
Wild Type ◽  
Leader Protein ◽  
16S Rna

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

1991 ◽  
Vol 11 (8) ◽  
pp. 4253-4265
Author(s):  
H G Wang ◽  
G Draetta ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Physical Interaction ◽  
Resting Cells ◽  
Wild Type ◽  
Quiescent Cells ◽  
Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

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