Part Two of BMB 400

1
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) Cover all but
Cellular control of replication pages 322-326,
question 6.11, 6.18, 6.19 Mutation and Repair
Chapter 7 Class Oct 09 Restrict coverage of
mutagenesis to types of mutations and UV
damage. Not cover these topics on pages
338-348 Errors in Replication Chemical
modification by oxidation Chemical modification
by alkylation Chemicals that cause
deletions Ionizing radiation Cover all repair
mechanisms Not 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.
2
Rest of Part Two
Recombination Chapter 8 Classes October 11,
16 Transposition Chapter 9 Class October
18 Exam October 21
3
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

4
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.

5
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.

6
Primosome has many proteins
Pre-priming complex
Protein gene function PriA priA
helicase, 3' to 5' movement, site
recognition PriB priB PriC priC DnaT
dnaT needed to add DnaB-DnaC complex to
preprimosome DnaC dnaC forms complex with
DnaB DnaB dnaB helicase, 5' to 3' movement,
is a hexamer DNA dependent ATPase.
Primase DnaG
7
Assay for assembly and migration of the primosome
Convert single stranded (ss) fX174 to duplex,
replicative form (RF)
8
Steps in priming and synthesis
9
Activities of DnaB and PriA in replisome
Sewing machine model
10
Control of DNA replication

• Replicon
• Origins and terminators
• Solutions to the end problem (telomeres)
• Cellular control mechanisms

11
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

12
Replicon unit that controls replication
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
13
Theta-form replication intermediates visualized
in EM for polyoma virus
B. Hirt
14
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 have the most
radioactivity.
15
2-D gels map number position of replication
origins
16
Positions of oriC and ter in E. coli
Replication fork 2
17
Structure of oriC
13
13
13
9
9
9
9

• 245 bp long
• 4 copies of a 9 bp repeat
• 3 copies of a 13 bp repeat
• 11 GATC motifs

1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG
TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA
ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA 121
AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA
TCTCTTATTA GGATCGCACT 181 GCCCTGTGGA TAACAAGGAT
CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT 241
GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA
GAATGAGGGG TTATACACAA 301 CTCAAAAACT GAACAACAGT
TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC 361
AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT
TGAGTAAATT AACCCACGAT
18
Initiation at oriC Model
19
Termination and resolution
20
Regulation of replication by methylation
m
m
G A T C
G A T C
C T A G
C T A G
m
m
G A T C
methylate
replicate
C T A G
m
(lags
m
G A T C
G A T C
behind
replication)
C T A G
C T A G
m
m
dam
methylase
Hemimethylated
Fully methylated
Fully methylated
Will not replicate
Will replicate
Will replicate
21
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