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潘学峰 [Xuefeng Pan]
Blog :http://blog.sciencenet.cn/u/duke01361
Email:xuefengpancam@aliyun.com
Organization:Hebei University, Beijing Institute of Technology and Baoding Great Wall Clinical
Academic title:Professor and TEO(Technical Ex. Officer)
[Academic experience]

Main Research Areas

  • The Molecular Mechanism of DNA Replication, Damage/Repair and Recombination,
    since year1993
  • Interactions of Important protein molecules within Cell,
    since year 2005
  • The Cellular and Molecular Basis of TCM Pharmacology,
    since year2011
  • Molecular biology and genetic engineering of Bacillus (Bacillus subtilis,since year 1990

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Slobodan Jergic, Nicholas P Horan, Mohamed M Elshenawy, Claire E Mason, Thitima Urathamakul, Kiyoshi Ozawa, Andrew Robinson, Joris M H Goudsmits, Yao Wang, Xuefeng Pan, Jennifer L Beck, Antoine M van Oijen, Thomas Huber, Samir M Hamdan, Nicholas E Dixon
EMBO J (2013), PMID 23435564

Processive DNA synthesis by the αɛθ core of the Escherichia coli Pol III replicase requires it to be bound to the β(2) clamp via a site in the α polymerase subunit. How the ɛ proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ɛ. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in ɛ that, in conjunction with the site in α, maintains a closed state of the αɛθ-β(2) replicase in the polymerization mode of DNA synthesis. The ɛ-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

 DOI: 10.1038/emboj.2012.347

 

 6. Determination of the reopening temperature of a DNA hairpin structure in vitro. Pan, Xuefeng. European Journal of Biochemistry vol. 271 issue 18 September 2004. p. 3665-3670

  A novel method, based upon primer extension, has been developed for measuring… (more)

Keywords: DNA hairpin; non-B DNA secondary structure; primer extension; reopening temperature; Tm

DOI: 10.1111/j.1432-1033.2004.04301.x. ISSN: 0014-2956.

More Like This

7. Visualization of protein RecR in Escherichia coli by fluorescent labelling. Qiu, Jiefang; Pan, Xuefeng. Progress in Natural Science vol. 19 issue 11 November 10, 2009. p. 1545-1551

 RecR protein, a functional equivalent of Rad52 in eukaryotes, plays a critical role… (more)

Keywords: RecR; YFP; DNA Repair; Homologous recombination; E. coli

DOI: 10.1016/j.pnsc.2009.07.003. ISSN: 1002-0071.

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8. Effects of degU32(Hy), degQa and degR Pleiotropic Regulatory Genes on the Growth and Protease Fermentation of Bacillus Subtilis Ki-2-132. PAN, Xue-Feng. Acta Genetica Sinica vol. 33 issue 4 April, 2006. p. 373-380

  Effects of degU32 (Hy), degR genes from Bacillus subtilis 168 and deg… (more)

Keywords: degU32 (Hy); deg Qa; degR; pleiotropic regulatory gene; Bacillus subtilis

DOI: 10.1016/S0379-4172(06)60063-2. ISSN: 0379-4172.

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9. Mechanism of Trinucleotide Repeats Instabilities: The Necessities of Repeat Non-B Secondary Structure Formation and the Roles of Cellular Trans-acting Factors. PAN, Xue-Feng. Acta Genetica Sinica vol. 33 issue 1 January, 2006. p. 1-11

  The mechanism underlying CAG·CTG, CGG·CCG and GAA·TTC trinucleotide repeats expansion and contraction… (more)

Keywords: trinucleotide repeats; expansion and contraction instability; fragility; human neurological-muscular disease; trans-acting factor

DOI: 10.1016/S0379-4172(06)60001-2. ISSN: 0379-4172.

 

[Distribution and evolution of simple repeats in the mtDNA D-loop in mammalian].

Jin-Pu JP Wei, Xue-Feng XF Pan, Hong-Quan HQ Li, Fei F Duan
Yi Chuan33(1):67-74 (2011), PMID 21377961

Simple sequence repeats (SSR) distribute extensively in genomes of all organisms, but the molecular mechanism underlined is poorly understood. In this study, we characterized distribution and biological significance of the simple repetitive DNA sequences in the D-loop region in mitochondria DNA of 256 mammal species, and classified the mammal carriers into three groups including 53 species with hexanucleotide repeats, 104 species with other types of simple repeats (>6 bp) and 99 species without any repeat sequences, respectively. Furthermore, we found that the hexanucleotide repeats dispersed significantly in the interval space between CSB1 and CSB2, while other repeats dispersed mainly in the termination region, central conserved region and the conserve sequence block (CSB) regions. In addition, comparison on the base composition and the DNA contexts of the central conserved region, CSB1, CSB2, and CSB3 revealed a lack of significant differences in similarity among different species with or without repeat sequences. Moreover, a phylogenetic analysis with 256 mammal species using N-J method suggested loss of the repeat sequences in mammals in evolution.

 

1. PLoS One. 2010 Dec 9;5(12):e14271. doi: 10.1371/journal.pone.0014271.

Transcription of AAT•ATT triplet repeats in Escherichia coli is silenced by H-NS 
and IS1E transposition.

Pan X, Liao Y, Liu Y, Chang P, Liao L, Yang L, Li H.

School of Life Science, Beijing Institute of Technology, Beijing, China.
xuefengpancam@yahoo.com.cn

BACKGROUND: The trinucleotide repeats AAT•ATT are simple DNA sequences that
potentially form different types of non-B DNA secondary structures and cause
genomic instabilities in vivo.
METHODOLOGY AND PRINCIPAL FINDINGS: The molecular mechanism underlying the
maintenance of a 24-triplet AAT•ATT repeat was examined in E. coli by cloning the
repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.
coli genome. Either the AAT or the ATT strand acted as lagging strand template in
a replication fork. Propagations of the repeats in either orientation on plasmids
did not affect colony morphology when triplet repeat transcription using the lacZ
promoter was repressed either by supplementing LacI(Q)in trans or by adding
glucose into the medium. In contrast, transparent colonies were formed by
inducing transcription of the repeats, suggesting that transcription of AAT•ATT
repeats was toxic to cell growth. Meanwhile, significant IS1E transposition
events were observed both into the triplet repeats region proximal to the
promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region 
itself. Transposition reversed the transparent colony phenotype back into
healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT
repeat in either orientation on plasmids did not produce significant changes in
cell morphology and did not promote IS1E transposition events. We further found
that a role of IS1E transposition into plasmids was to inhibit transcription
through the repeats, which was influenced by the presence of the H-NS protein,
but not of its paralogue StpA.
CONCLUSIONS AND SIGNIFICANCE: Our findings thus suggest that the longer AAT•ATT
triplet repeats in E. coli become vulnerable after transcription. H-NS and its
facilitated IS1E transposition can silence long triplet repeats transcription and
preserve cell growth and survival.

PMCID: PMC3000339
PMID: 21151567  [PubMed - indexed for MEDLINE]


2. DNA Repair (Amst). 2009 Nov 2;8(11):1321-7. doi: 10.1016/j.dnarep.2009.08.001.
Epub 2009 Sep 4.

The roles of SbcCD and RNaseE in the transcription of GAA x TTC repeats in
Escherichia coli.

Pan X, Ding Y, Shi L.

School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
xuefengpancam@yahoo.com.cn

Expansion of GAA x TTC repeats in FXN gene is associated with decreased frataxin 
production in Frederich's ataxia patients. To study this effect, we have
engineered a set of GAA x TTC repeats in the EcoRI site of lacZ gene of plasmid
pUC18 as part of the transcription template of the lacZ gene, while keeping its
ORF unchanged. The effects of the GAA x TTC repeats on the lacZ expression were
investigated in Escherichia coli JM83 and its mutants deficiency in RNA
processing, homologous recombination and DNA repair. We found that transcriptions
of the GAA strand with different sizes and organizations displayed normal
alpha-complementation when RNase E was functional. By contrast, transcriptions of
TTC repeats containing more than 13 triplets failed to support
alpha-complementation, showing RNase-independent but length-dependent effects of 
TTC repeats on lacZ expression. In addition, we also found that functions of
SbcCD, a DNA structure specific nuclease, were needed in the RNase E-dependent
lacZ expression of the GAA repeats. These suggested that processing of DNA and
RNA is essential to the transcription of the repeats-carrying gene in vivo.

PMID: 19733517  [PubMed - indexed for MEDLINE]


3. J Genet Genomics. 2006 Jan;33(1):1-11.

Mechanism of trinucleotide repeats instabilities: the necessities of repeat non-B
secondary structure formation and the roles of cellular trans-acting factors.

Pan XF.

Institute of Cell and Molecular Biology, Edinburgh University, Edinburgh EH9 3JR,
UK. xuefengpancam@yahoo.com.cn

The mechanism underlying CAG.CTG CGG.CCG and GAA.TTC trinucleotide repeats
expansion and contraction instabilities has not been clearly understood.
Investigations in vitro have demonstrated that the disease causing repeats are
capable of adopting non-B secondary structures that mediate repeats expansion.
However, in vivo, similar observations have not been easily made so far.
Investigations on the non-B secondary structure formation using E.coli, yeast etc
cannot simulate the suggested repeats expansion instability. These could leave a 
space to infer a disassociation of the suggested repeats non-B secondary
structure formation and the repeats expansion in vivo. Although longer
trinucleotide repeats may be theoretically easier to form non-B DNA secondary
structures in replication or in post-replication, however such non-B secondary
structures are likely to cause repeat fragility rather than repeat expansion. In 
fact, repeat expansion as seen in patients may not necessarily require
trinucleotide repeats to form non-B secondary structures, instead the repeat
expansions can be produced through a RNA transcription-stimulated local repeat
DNA replication and a subsequent DNA rearrangement.

PMID: 16450581  [PubMed - indexed for MEDLINE]


4. Microbiol Res. 2004;159(2):97-102.

Two-triplet CGA repeats impede DNA replication in bacteriophage M13 in
Escherichia coli.

Pan X.

Institute of Microbiology, Chinese Academy of Sciences, Academy of Science,
Beijing 100080, China. X.Pan@staffemail.ac.uk

Expansion and contraction instabilities associated with CAG, CGG, GAA and CGA
(GAC) repeats propagation cause more than a dozen human genetic diseases and
cancers. In this work, the propagation behavior of a bacteriophage M13 carrying a
calf prochymosin cDNA fragment with a (CGA)2 repeat in a small hairpin forming
region is reported. Such a M13 derivative when propagated in Escherichia coli,
produces small plaques by decreasing phage yield and also mitigates the
inhibition on host cell growth, compared to those control bacteriophages either
containing a "CTGCTA" sequence or wildtype, suggesting that CGA2 repeat impedes
DNA replication in vivo. Moreover, an increased internal free energy is found
associated with (CGA)2 sequence compared to those "CTGCTA" and wildtype, which
ruled out a possibility of CGA2 repeat effects on propagation is through
influencing the hairpin structure formation.

PMID: 15293942  [PubMed - indexed for MEDLINE]


5. Nucleic Acids Res. 2000 Aug 15;28(16):3178-84.

The roles of mutS, sbcCD and recA in the propagation of TGG repeats in
Escherichia coli.

Pan X, Leach DR.

Institute of Cell and Molecular Biology, The University of Edinburgh, Kings
Buildings, Edinburgh EH9 3JR, UK.

A 24 triplet TGG.CCA repeat array shows length- and orientation-dependent
propagation when present in the plasmid pUC18. When TGG(24) is present as
template for leading-strand synthesis, plasmid recovery is normal in all strains 
tested. However, when it acts as template for lagging-strand synthesis, plasmid
propagation is seriously compromised. Plasmids carrying deletions in the 5' side 
of this sequence can be isolated and products carrying 15 TGG triplets do not
significantly interfere with plasmid propagation. Mutations in sbcCD, mutS and
recA significantly improve the recovery of plasmids with TGG(24) on the
lagging-strand template. These findings suggest that TGG(24) can fold into a
structure that can interfere with DNA replication in vivo but that TGG(15)
cannot. Furthermore, since the presence of the MutS and SbcCD proteins are
required for propagation interference, it is likely that stabilisation of
mismatched base pairs and secondary structure cleavage are implicated. In
contrast, there is no correlation of triplet repeat expansion and deletion
instability with predicted DNA folding. These results argue for a dissociation of
the factors affecting DNA fragility from those affecting trinucleotide repeat
expansion-contraction instability.

PMCID: PMC108438
PMID: 10931934  [PubMed - indexed for MEDLINE]