Physical and functional interactions of human DNA polymerase {α-primase [alpha-primase] and replication protein A [Elektronische Ressource] / von Kamran Ali Mirza

Hp Nasheuer

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2005
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Physical and Functional Interactions of Human DNA
Polymerase α-primase and Replication Protein A










Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr.rer.nat.)













vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena


Von
(M.Phil Biotechnology) Kamran Ali Mirza
Geboren am 05.03.1972
in Lahore, Pakistan

















Gutachter:
1....................................................................................................................................................

2....................................................................................................................................................

3....................................................................................................................................................

Tag der Doktorprüfung................................................................................................................

Tag der öffentlichen Verteidigung.............................................................................................. Index i




1 Introduction ......................................................................................................................1
1.1 Cell cycle and DNA replication................................................................................... 1
1.2 DNA polymerase α-primase........................................................................................ 6
1.2.1 Structure of pol-prim complex................................................................................................ 7
1.2.2 Reaction mechanism of pol-prim complex at the replication fork........................................ 10
1.2.3 Regulation of pol-prim during the cell cycle ........................................................................ 11
1.3 Replication protein A................................................................................................. 13
2 Materials and Methods...................................................................................................16
2.1 Materials ..................................................................................................................... 16
2.1.1 Cell lines.......... 16
2.1.2 Bacterial strains .................................................................................................................... 16
2.1.3 DNA (Plasmids, Constructs) ............................................................................................... 16
2.1.4 Oligonucleotides................................................................................................................... 17
2.1.5 Restriction enzymes.............................................................................................................. 19
2.1.6 Proteins................................................................................................................................. 19
2.1.7 Antibodies........ 19
2.1.8 Protein-molecular weight marker ......................................................................................... 20
2.1.9 Apparatus.............................................................................................................................. 21
2.1.10 Chemicals.. 22
2.1.11 Computer programs ......................................................................................................... 23
2.2 Methods 24
2.2.1 Cell culture ........................................................................................................................... 24
2.2.2 Preparation of crude cell extracts.......................................................................................... 24
2.2.3 Determination of protein concentration................................................................................ 24
2.2.4 Immunoprecipitation ............................................................................................................ 24
2.2.5 Transfection....... 25
2.2.6 Labeling of oligonucleotides ................................................................................................ 26
2.2.7 Electrophoretic mobility shift assay ..................................................................................... 27
2.2.8 SDS polyacrylamide gel electrophoresis .............................................................................. 27
R2.2.9 Coomassie Brilliant Blue staining of protein ...................................................................... 28
2.2.10 Western blotting............................................................................................................... 28
2.2.11 Purification of recombinant human primase .................................................................... 31
2.2.12 Primase assay...................................................................................................................32
2.2.13 DNA polymerase assay.................................................................................................... 32
3 Results.............................................................................................................................33
3.1 Expression and purification of recombinant human primase ............................... 33
3.2 Primase binding to M13-ssDNA ............................................................................... 35
3.3 Primase and its subunits bind to oligonucleotides .................................................. 35
3.4 CTp58 binds to oligonucleotides 41
3.5 Inhibition of primase activity in the presence of oligonucleotides......................... 43
3.6 Replication protein A (RPA) and primase co-operate in their binding ................ 45
to DNA........................................................................................................................ 45
3.7 Iron effects primase binding to different oligonucleotides..................................... 48
Index ii




3.8 Addition of iron III to iron-depleted protein reconstitutes DNA-binding ............ 49
3.9 Primase assay with iron-containing and iron-depleted primase............................ 50
3.10 Localization of transfected (Pol α-GFP) fusion proteins in fixed and .................. 53
living cells.................................................................................................................... 53
3.11 Immunoprecipitation of recombinant p180 (T7-tag) in human cells with............ 54
T7-tag antibody .......................................................................................................... 54
3.12 Recombinant DNA polymerase α subunits form complex with each.................... 55
other............................................................................................................................ 55
3.13 Association of endogenous primase and ectopically expressed p180..................... 55
3.14 Association of replication protein A with DNA polymerase α-primase................ 57
4 Discussion.......................................................................................................................60
4.1 Expression and purification of recombinant human primase in E. coli ............... 60
4.2 Characterization of primase binding to DNA ......................................................... 61
4.2.1 Primase binding to single stranded DNA (M13-ssDNA) ..................................................... 61
4.2.2 Primase binding with different structured oligonucleotides ................................................. 63
4.3 Replication protein A (RPA) and primase co-operate in their binding to DNA .. 64
4.4 Iron effects primase binding to DNA ....................................................................... 65
4.5 Localization of DNA polymerase α .......................................................................... 66
4.6 Pol-prim interacting partners ................................................................................... 67
5 Summary.........................................................................................................................68
6 References.......................................................................................................................71

Abbreviations i




Abbreviations


aa Amino acids
APS Ammonium per sulphate
ATM Ataxia telangiectasia protein M
ATPAdenosinetriphosphate
BER Base excision repair
bp Base pair
BSA Bovine serum albumin
°CDegreecelsius
CdKCyclin-dependent kinase
CTp58 C-terminus of p58
Da Dalton
DDKDbf-dependentkinase
DNADeoxyribonucleic acid
dsDouble-stranded
EDTA Ethylenediamine tetraacetic acid
EMSA Electrophoretic mobility shift assay
Fe Iron
gGram
hr.Hour
hrs.Hours
HRP Horse-radish-peroxidase
IB Immuno blotting (Western blotting)