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Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/15/05

Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/15/05

CHAPTER 8: The replication of DNA Molecular Biology Course CHAPTER 8: The replication of DNA

Teaching Arrangement Watch animation-Understand replication CHAPTER 8 The replication of DNA Teaching Arrangement Watch animation-Understand replication Go through structural tutorial-Experience the BEAUTY of the nature QA-comprehensive understanding of Chapter 8 Summary and highlight Key points

General Detailed The Chemistry of DNA Synthesis CHAPTER 8 The replication of DNA The Chemistry of DNA Synthesis The Mechanism of DNA Polymerase The Replication Fork The Specialization of DNA Polymerases DNA Synthesis at the Replication Fork Initiation of DNA Replication Binding and Unwinding Finishing Replication General Detailed

CHAPTER 8 The replication of DNA The first part describes the basic chemistry of DNA synthesis and the function of the DNA polymerase

CHAPTER 8 The replication of DNA The Chemistry of DNA DNA synthesis requires deoxynucleoside triphosphates and a primer:template junction DNA is synthesized by extending the 3’ end of the primer Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis 3/18/05

Figure 8-3 Substrate required for DNA synthesis

The mechanism of DNA Polymerase (Pol) CHAPTER 8 The replication of DNA The mechanism of DNA Polymerase (Pol) 3/18/05

DNA Pol use a single active site to catalyze DNA synthesis The mechanism of DNA Pol A single site to catalyze the addition of any of the four dNTPs. Recognition of different dNTP by monitoring the ability of incoming dNTP in forming A-T and G-C base pairs; incorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity). 3/18/05

Figure 8-3

Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site. The mechanism of DNA Pol Figure 8-4 3/18/05

DNA Pol resemble a hand that grips the primer-template junction The mechanism of DNA Pol Schematic drawing T7 DNA pol Figure 8-5

Thumb Fingers Palm Figure 8-8

DNA Polymerase-palm domain*** Catalytic sites for addition and removal of dNTPs. Binds to two metal ions that alter the chemical environment around the catalytic site. (how?) 3/18/05

DNA Polymerase-finger domain Binds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysis Bends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotide Stabilization of the pyrophosphate

DNA Polymerase-thumb domain Not directly involved in catalysis Interacts with the synthesized DNA to maintain correct position of the primer and the active site, and to maintain a strong association between DNA Pol and its substrate. 3/18/05

DNA Pol are processive enzymes The mechanism of DNA Pol Processivity is a characteristic of enzymes that operate on polymeric substrates. The processivity of DNA Pol is the average number of nucleotides added each time the enzyme binds a primer:template junction (a few~50,000).

The rate of DNA synthesis is closely related to the polymerase processivity, because the rate-limiting step is the initial binding of polymerase to the primer-template junction.

Figure 8-9

Exonucleases proofread newly synthesized DNA The mechanism of DNA Pol The occasional flicking of the bases into “wrong” tautomeric form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease, a part of the DNA polymerase. How does the exonucleases work? Kinetic selectivity

Figure 8-10

CHAPTER 8 The replication of DNA The second part describes how the synthesis of DNA occurs in the context of an intact chromosome at replication forks. An array of proteins are required to prepare DNA replication at these sites.

CHAPTER 8 The replication of DNA The replication fork The junction between the newly separated template strands and the unreplicated duplex DNA 3/18/05

Both strands of DNA are synthesized together at the replication fork. Leading strand Okazaki fragment Replication fork Lagging strand Figure 8-11

The initiation of a new strand of DNA require an RNA primer The replication fork Primase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence. DNA Pol can extend both RNA and DNA primers annealed to DNA template

RNA primers must be removed to complete DNA replication The replication fork A joint efforts of RNase H, DNA polymerase & DNA ligase Figure 8-12

Topoisomerase removes supercoils produced by DNA unwinding at the replication fork Figure 8-15

DNA helicases unwind the double helix in advance of the replication fork Hexameric protein (see your CD) Figure 8-13

Single-stranded binding proteins (SSBs) stabilize single-stranded DNA The replication fork Cooperative binding Sequence-independent manner (electrostatic interactions) Figure 8-14

Replication fork enzymes extend the range of DNA polymerase substrate The replication fork DNA Pol can not accomplish replication without the help of other enzymes DNA helicase, SSB, primase, DNA topoisomerase

The specialization of DNA polymerases CHAPTER 8 The replication of DNA The specialization of DNA polymerases 3/18/05

DNA Pols are specialized for different roles in the cell The specialization of DNA pol Each organism has a distinct set of different DNA Pols Different organisms have different DNA Pols DNA Pol III holoenzyme: a protein complex responsible for E. coli genome replication DNA Pol I: removes RNA primers in E. coli

Polymerase switching: the process of replacing DNA Pola/primase with DNA Pold or DNA Pole. Table 8-2

Sliding clamps dramatically increase DNA polymerase activity The specialization of DNA pol Encircle the newly synthesized double-stranded DNA and the polymerase associated with the primer:template junction Ensures the rapid rebinding of DNA Pol to the same primer:template junction, and thus increases the processivity of Pol. Eukaryotic sliding DNA clamp is PCNA

Figure 8-17

Figure 8-19 Sliding DNA clamps are found across all organism and share a similar structure

Sliding clamps are opened and placed on DNA by clamp loaders The specialization of DNA pol Clamp loader is a special class of protein complex catalyzes the opening and placement of sliding clamps on the DNA, such a process occurs anytime a primer-template junction is present. Sliding clamps are only removed from the DNA once all the associated enzymes complete their function.

DNA synthesis at the replication fork CHAPTER 8 The replication of DNA DNA synthesis at the replication fork 3/18/05

At the replication, the leading strand and lagging strand are synthesized simultaneously. The biological relevance is listed in P205-206 To coordinate the replication of both strands, multiple DNA Pols function at the replication fork. DNA Pol III holoenzyme is such an example.

Figure 8-20 The composition of the DNA Pol III holoenzyme

Figure 8-21*** Trombone model

DNA synthesis at the replication fork Interactions between replication fork proteins form the E. coli replisome Replisome is established by protein-protein interactions DNA helicase & DNA Pol III holoenzyme, which is mediated by the clamp loader and stimulates the activity of the helicase (10-fold) DNA helicase & primase, which is relatively week and strongly stimulates the primase function (1000-fold). This interaction is important for regulation the length of Okazaki fragments.

DNA Pol III holoenzyme, helicase and primase interact with each other to form replisome, a finely tuned factory for DNA synthesis with the activity of each protein is highly coordinated.

CHAPTER 8 The replication of DNA The third part focuses on the initiation and termination of DNA replication. Note that DNA replication is tightly controlled in all cells and initiation is the step for regulation.

Initiation of DNA replication CHAPTER 8 The replication of DNA Initiation of DNA replication 3/18/05

Initiation of DNA replication Specific genomic DNA sequences direct the initiation of DNA replication Origins of replication, the sites at which DNA unwinding and initiation of replication occur. 3/18/05

The replicon model of replication initiation Initiation of DNA replication The replicon model of replication initiation Proposed by Jacob and Brenner in 1963 All the DNA replicated from a particular origin is a replicon Two components, replicator and initiator, control the initiation of replication 3/18/05

Replicator: the entire site of cis-acting DNA sequences sufficient to direct the initiation of DNA replication Initiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replication Figure 8-23

Initiation of DNA replication Replicator sequences include initiator binding sites and easily unwound DNA 3/18/05

CHAPTER 8 The replication of DNA Binding and Unwinding: origin selection and activation by the initiator protein 3/18/05

Three different functions of initiator protein: (1) binds to replicator, (2) distorts/unwinds a region of DNA, (3) interacts with and recruits additional replication factors DnaA in E. coli (all 3 functions), origin recognition complex (ORC) in eukaryotes (functions 1 & 3)

Binding and unwinding Protein-protein and protein-DNA interactions direct the initiation process DnaA recruits the DNA helicase DnaB and the helicase loader DnaC DnaB interacts with primase to initiate RNA primer synthesis, see replisome part for more details. 3/18/05

Figure 8-26*

Binding and unwinding Eukaryotic chromosome are replicated exactly once per cell cycle, which is critical for these organims 3/18/05

Pre-replicative complex (pre-RC) formation directs the initiation of replication in eukaryotes Binding and unwinding Initiation in eukaryotes requires two distinct steps Replicator selection: the process of identifying sequences for replication initiation (G1 phase), which is mediated by the formation of pre-RCs at the replicator region.

Figure 8-29 pre-RC formation

Origin activation: pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase. Figure 8-30 pre-RC activation & assembly of the replication fork in eukaryotes

Binding and unwinding Pre-RC formation and activation is regulated to allow only a single round of replication during each cell cycle. Only one opportunity for pre-RCs to form, and only one opportunity for pre-RC activation.

Figure 8-31 Effect of Cdk activity on pre-RC formation and activation

Figure 8-32 Cell cycle regulation of Cdk activity and pre-RC formatin

Finishing replication CHAPTER 8 The replication of DNA Finishing replication 3/18/05

Type II topoisomerases are required to separate daughter DNA molecules Finishing replication

The End replication problem (Figure 8-34) Lagging strand synthesis is unable to copy the extreme ends of the linear chromosome Finishing replication The End replication problem (Figure 8-34)

How telomerase works? (Figure 8-36 Telomerase is a novel DNA polymerase that does not require an exogenous template Finishing replication How telomerase works? (Figure 8-36 How the end problem is eventually resolved? (Figure 8-37)

T1:The Chemistry of DNA Synthesis T2: The Mechanism of DNA Polymerase CHAPTER 8 The replication of DNA T1:The Chemistry of DNA Synthesis T2: The Mechanism of DNA Polymerase T3: The Replication Fork （分） T4: The Specialization of DNA Polymerases （分） T5: DNA Synthesis at the Replication Fork （合） T6: Initiation of DNA Replication T7: Binding and Unwinding T8: Finishing Replication

重点 Completely understand 三个Animations CHAPTER 8 The replication of DNA 重点 Completely understand 三个Animations DNA polymerization (Topics 1 & 2) DNA replication (Topics 3－5) Action of Telomerase (Topic 8) 3/18/05

Topic 6-7: Initiation of DNA replication Topic 6-7: Initiation of DNA replication. 重点掌握(1) 概念origin of replication, replicator, initiator (DnaA & ORC) ,以及图8－23，25, 26; （2）How the eukaryotic chromosomes are ensured to replicate exactly once per cell cycle?

Homework Complete all the excises on your study CD. Enjoy it