part two of bmb 400
DESCRIPTION
Part Two of BMB 400. Enzymes needed for DNA replication: Chapter 5 Classes Sept 23, 25, Oct 02, 07 Covered entire chapter Origins, terminators and control of replication: Chapter 6 Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80 (questions 2.12-2.14) - PowerPoint PPT PresentationTRANSCRIPT
Part Two of BMB 400
Enzymes needed for DNA replication: Chapter 5Classes Sept 23, 25, Oct 02, 07Covered entire chapter
Origins, terminators and control of replication: Chapter 6Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80
(questions 2.12-2.14)Cover all but “Cellular control of replication” pages 322-326, question 6.11, 6.18, 6.19
Mutation and Repair: Chapter 7Class: Oct 09Restrict coverage of mutagenesis to types of mutations and UV damage.Not cover these topics on pages 338-348
Errors in ReplicationChemical modification by oxidationChemical modification by alkylationChemicals that cause deletionsIonizing radiation
Cover all repair mechanismsNot cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16.
[For questions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7.16 to see the damaged DNA to be repaired.]
Rest of Part Two
Recombination: Chapter 8Classes: October 11, 16
Transposition: Chapter 9Class October 18
Exam: October 21
October 07 class
• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:
2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling
Primase
• Synthesizes short oligonucleotides from which DNA polymerases can begin synthesis.– Combination of ribonucleotides and
deoxyribonucleotides
• Does not itself require a primer.
• E. coli primase is DnaG, 60 kDa
• Acts within a large primosome.
Primers made by DnaG
• Primers can be as short as 6 nt, as long as 60 nt.
• Can substitute dNTPs for rNTPs in all except 1st and 2nd positions– Make hybrid primers with dNMPs and rNMPs
interspersed.
• Primase binds to CTG– T serves as template for 1st nucleotide of
primer.
Primosome has many proteins
Protein gene functionPriA priA helicase, 3' to 5' movement, site recognitionPriB priBPriC priCDnaT dnaT needed to add DnaB-DnaC complex to preprimosomeDnaC dnaC forms complex with DnaBDnaB dnaB helicase, 5' to 3' movement, is a hexamer
DNA dependent ATPase.
Pre-priming complex:
Primase = DnaG
Assay for assembly and migration of the primosome
+ +-
ss RF
Convert single stranded (ss) X174 to duplex, replicative form (RF)
Steps in priming and synthesis
PriA
PriBPriCDnaT
DnaCDnaB
primer assembly site
ADP + Pi
ATP
ATP
ADP + Pipreprimingcomplex
SS DNAcoated with SSB
recognition step
primosomeNTPs
PPi's
primer synthesis
DnaG
(primase)
Activities of DnaB and PriA in replisome
“Sewing machine” model
Control of DNA replication
Replicon
Origins and terminators
Solutions to the “end problem” (telomeres)
Cellular control mechanisms
Oct 07 Class
• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:
2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling
Replicon = unit that controls replication
InitiatorReplicator Replicator
Initiator
+
E. coli ori C DnaA
Yeast ARS ,
an autonomously
replicating sequence
ORC (the
origin recognition complex)
+ ABF1 ( ARS binding factor 1)
Complex of
Replicator +
initiator allows
replication to
begin
duplex
DNA
Replicator: cis-acting DNA sequence required for initiation; defined genetically
Origin: site at which DNA replication initiates; defined biochemically
Initiator: protein needed for initiation, acts in trans
Theta-form replication
intermediates visualized in
EM for polyoma virus
B. Hirt
Labeling of completed DNA molecules can map replication origins
Dana and Natahans, 1972, PNAS: map the replication origin of SV40 by labeling replicating molecules for increasing periods of time, isolating complete molecules, digesting with Hind restriction endonucleases, and determining which fragments havethe most radioactivity.
A
C
D
E
K
FJ
GB
I
H
Physical map of the SV40 DNA fragmentsproduced by cleavage with H. influenzarestriction endonucleases
2-D gels: map number & position of replication origins1st dimension separates by size
2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc" "Bubble-arc" Related to distance from ori to end of fragment.
Simple Y Bubble Double Y Asymmetric
Fragmentsize doublesduringreplication.
Positions of oriC and ter in E. coliForks meet and terminate in this approx. 100 kb region
and are 23 bp binding sites for Tus, a "contra-helicase."terC and terB
block progress of Fork 2
terD and terAblock progress of Fork 1
E. coli chromosome
oriC
Replication fork 1
245 bp
Replication fork 2
Structure of oriC
• 245 bp long– 4 copies of a 9 bp repeat– 3 copies of a 13 bp repeat– 11 GATC motifs
13 1313 9 9 99
1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA121 AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA TCTCTTATTA GGATCGCACT181 GCCCTGTGGA TAACAAGGAT CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT241 GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA GAATGAGGGG TTATACACAA301 CTCAAAAACT GAACAACAGT TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC361 AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT TGAGTAAATT AACCCACGAT
Initiation at oriC: Model
Termination and
resolution
Regulation of replication by methylation
G A T C
C T A G
m
m
G A T C
C T A G
m
G A T C
C T A Gm
G A T C
C T A G
m
m
G A T C
C T A G
m
m
replicate methylate(lags behind replication)
Fully methylated Hemimethylated
dam methylase
Fully methylated
Will replicate Will not replicate Will replicate
Oct 07 Class
• Finish replication enzymes: 2_2_repl_enzy2.pdf– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf• Topological problems in replication:
2_4_telom_topo_reg.pdf– Telomerase– Topoisomerases– DNA supercoiling