DNA Replication The basic rules for DNA replication DNA Polymerases Initiation of replication DNA synthesis at the replication fork Termination of replication Regulation of re-initiation Other modes of DNA replication
Initiation of replication Common features of replication origins Common events of initiation Priming
The replicon model of replication initiation: (proposed by F. Jacob, S. Brenner and J. Cuzin, 1963) All the DNA replicated from a particular origin as a replicon. Binding of the initiator to the replicator stimulates initiation of replication.
Replicator Initiator Replicator: the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of DNA replication (*the origin of replication is part of replicator) Initiator: the DNA-binding protein that specifically recognizes a DNA element in the replicator and activates the initiation of replication
Priming and DNA synthesis Easily melted region Initiator binding site Replicator (Origin) DNA binding Initiator DNA unwinding The functions of initiator Protein recruitment Priming and DNA synthesis
E. coli oriC DnaA DnaB/DnaC DNA binding DNA unwinding Protein recruitment DnaB/DnaC Priming and DNA synthesis
oriC : the origin of replication in E. coli Figure 14.26 Easily melted (AT rich) Initiator (DnaA) binding site
oriC: L, M, R repeats (13 bp) 1~4 repeats (9 bp) GATCTNTTNTTTT Consensus sequence Consensus sequence GATCTNTTNTTTT CTAGANAANAAAA TTATNCANA AATANGTNT
Origin recognition complex (ORC) The replicator (origin) of S. cerevisiae: ARS (Autonomously replicating sequence) ~ 150 bp Origin recognition complex (ORC) (Initiation complex) ACS: ARS consensus sequence 圖引用自:Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17
Mutations in B elements reduce origin function. Figure 13.20 Mutation in ARS Mutations in B elements reduce origin function. Mutations in core consensus abolish origin function.
E. coli oriC DnaA DnaB/DnaC DNA binding DNA unwinding Protein recruitment DnaB/DnaC Priming and DNA synthesis
DnaA.ATP DNA helicase (DnaB) DNA helicase Loader (DnaB) Figure 14.27 Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-26.
(histone like protein) DnaAATP HU + ATP (histone like protein) DNA bending Super-helical tension Strand separation Binding of DnaA, plus the basic proteins, bends the DNA rather sharply and creates negative super-helical tension. In turn this tension causes DNA unwinding in the 13 bp regions.
DNA helicase (DnaB) DNA helicase Loader (DnaB)
Figure 14.10
bind to the ssDNA, preventing it Two types of function are needed to convert dsDNA to the single-stranded state: Helicases separate the strands of DNA, usually using the hydrolysis of ATP to provide the necessay energy 2. Single-strand binding proteins bind to the ssDNA, preventing it from reforming the duplex state
Single-strand binding proteins DNA helicase (DnaB) DNA helicase Loader (DnaB) Single-strand binding proteins Primase
For DNA replication, a primase is required to catalyze the synthesis of RNA primer. Primase in E. coli: An RNA polymerase Synthesizing short stretches of RNA Encoded by the dnaG gene
Figure 14.14
Protein required to initiate replication at the E. coli origin: Summary Protein required to initiate replication at the E. coli origin: Protein Function DnaA protein Recognizes origin sequence; open duplex at specific sites in origin DnaB protein (helicase) Unwinds DNA DnaC protein Required for DnaB binding at origin HU DNA bending protein; stimulates initiation Primase (DnaG protein) Synthesizes RNA primers Single-strand DNA-binding protein (SSB) Binds single-strand DNA DNA gyrase (DNA topoisomerase) Relieves torsional strain generated by DNA unwinding Dam methylase Methylates 5’-GATC-3’ sequences at oriC The timing of replication initiation is affected by DNA methylation and interactions with the bacterial plasma membrane. The oriC region of E.coli is highly enriched in GATC sequences, containing 11 of them in its 245 bp.( See below for regulation of re-initiation).
DNA Replication The basic rules for DNA replication DNA Polymerases Initiation of replication DNA synthesis at the replication fork Termination of replication Regulation of re-initiation Other modes of DNA replication
DNA synthesis at the replication fork Proteins at the replication forks Coordinating synthesis of the lagging and leading strands in E. coli in eukaryotic cells
DNA replication is semidiscontinuous. Figure 14.9 Okazaki fragments 1000-2000 nt in prokaryotes 100-400 nt in eukaryotes
DNA replicase (DNA polymerase) Proteins required at the replication forks: Topoisomerase Helicase Primase SSB DNA replicase (DNA polymerase) Primosome Primer removal enzyme DNA ligase *SSB: Single-strand binding proteins
Different replicase units are required to synthesize the leading and lagging strands. In E. coli both units contain the same catalytic subunit of DNA Pol III. In other organisms, different catalytic subunits may be required for each strand.
The helicase creating the replication fork is connected to two DNA polymerase catalytic subunits. Each polymerase catalytic subunit is held on DNA by a sliding clamp. Figure 14.19
synthesizes the lagging strand dissociates at the end of Okazaki The polymerase that synthesizes the lagging strand dissociates at the end of Okazaki fragment and then reassociates with a primer in the single- stranded template loop to synthesize the next fragment. The polymerase that synthesizes the leading strand moves continuously. Figure 14.19
In E. coli: DnaB DNA Pol III holoenzyme DnaG
E. coli DNA Polymerase III holoenzyme g d d’ e q a t b c Based on Figure 14.17
a a t t c y g d d’ Clamp loader b Sliding clamp b Sliding clamp e e q proofreading e e polymerization polymerization a a q q Core enzyme t t Core enzyme dimerization
y d’ c y d’ c g d g d b y d’ c g d b b y d’ Pi c g d b b Clamp loader ATP ATP c y g d d’ b b b Sliding clamp ADP c y g d d’ b b ATP c y g d d’ Pi ATP hydrolysis b b
Processivity Core enzyme: 10 ~ 15 Holoenzyme: >500000 b converts Pol III from a distributive enzyme to a highly processive enzyme. Figure 14.18
a a t t y d’ c b g d b e e q q DnaB (helicase) Leading strand synthesis Lagging strand synthesis e e a a q q t t t subunits maintain dimeric structure of Pol III and interact with DnaB DnaB (helicase)
DnaB (helicase) is responsible for forward movement at the replication fork. Each catalytic core of polymerase III synthesizes a daughter strand. Figure 14.20
What happens to the loop when the Okazaki fragment is completed? Figure 14.21
1 4 2 3 Initiation of Okazaki fragment Reassociation of b clamp 5. Reassociation of core 1 4 Initiation of Okazaki fragment Reassociation of b clamp 2 3 Termination of Okazaki fragment Dissociation of core and b clamp Figure 14.20
Each Okazaki fragment is synthesized as a discrete unit. Lagging strand Primase synthesizes RNA primer. Leading strand DNA Pol III extends primer into Okazaki fragment. Next Okazaki fragment is synthesized.
Okazaki fragments are linked together. 3’ 5’ 3’ 5’ RNA primer DNA Pol I uses nick translation to replace RNA primer with DNA. Ligase seals the nick. Figure 14.22
5’3’ Exonuclease activity of DNA Pol I: Figure 14.5
Mechanism of the DNA ligase reaction P O O- Ribose Adenine R AMP NAD+ (R = NMN) or ATP (R = PPi) Ligase NH3 + + Adenylylation of DNA ligase 1 Ligase NH2 + P O O- Ribose Adenine + NMN (or PPi)
2 * 3 Activation of 5’ phosphate in nick O + Ligase NH2 P O Ribose Figure 14.23 O 2 + Ligase NH2 P O Ribose Adenine O- Activation of 5’ phosphate in nick Ligase NH3 + * 3
Adenine Ribose O O- 3 P O O O- The 3’-hydroxyl group attacks the phosphate and displaces AMP, producing a phosphodiester bond. P O O HO ●● 3’
Eukaryotic cell Pold/e Pold PCNA Pola/primase (RFC)
Eukaryotes have many DNA polymerases. Figure 14.24
a + - d e Eukaryotic DNA polymerases for replication in nucleus: DNA Primase activity Processivity Proof-reading Function a + moderate - Primer synthesis d High Leading/lagging? strand e lagging strand synthesis
DNA polymerase a (Pola/primase): 2 subunits: Pol a DNA synthesis 2 subunits: primase RNA synthesis 3’ 5’ 5’ OH 3’ RNA DNA (iDNA) ~ 10 bp 20-30 bp
DNA polymerase switching during eukaryotic DNA replication: DNA Pol a/ primase RNA primer synthesis by primase a P Watson, J. D. et al. (2004) Molecular Biology of the gene. 5th ed. CSHL Press. Fig. 8-16. RNA DNA synthesis by Pol a iDNA
iDNA Sliding clamp * Pola/primase DNA Pol e (or d)
* R-FC binds to the 3’ end of iDNA and displaces pol a/primase R-FC attracts PCNA PCNA binds pol d or e RF-C: Clamp loader PCNA: Sliding clamp
PCNA Proliferating cell nuclear antigen : (trimer) 圖引用自:Voet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-1
DNA topoisomerases are also required! Summary Proteins required at the replication forks: Figure 14.25 DNA topoisomerases are also required!
There are two ways to think of the relative motion of the DNA and replication machinery: The replication machinery moves along the DNA. (similar to a train moving along its track) 2. The DNA moves while the replication machinery is static. (similar to film moving into a movie projector)
The two replisomes of E. coli are linked together and tethered to one point on the bacterial inner membrane. Pol III holoenzyme Pol III holoenzyme Helicases (double-hexamers) 圖引用自:Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a
Cell elongates as replication continues Origin Cells divide Replication begins Chromosome Terminator Replisomes Chromosomes separate Origins separate Cell elongates as replication continues 圖引用自:Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-18a