Part Two of BMB 400 needed for DNA replication: Chapter 5 asses Sept 23, 25, Oct 02, 07 vered entire chapter terminators and control of replication: Chapter 6 asses Oct 04, 07. Note that supercoiling is from Chapter 2, pages 7 (questions 2.12-2.14) ver all but “Cellular control of replication” pages 322-326, questi 18, 6.19 and Repair: Chapter 7 ass: Oct 09 strict coverage of mutagenesis to types of mutations and UV damage. t cover these topics on pages 338-348 Errors in Replication Chemical modification by oxidation Chemical modification by alkylation Chemicals that cause deletions Ionizing radiation ver all repair mechanisms t cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16. estions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7 damaged DNA to be repaired.]
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 Presentation
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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.]
• 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)
• 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