Principle of DNA replication

Chen Liuh-yow Molecular Biology of the Cell, Chapter 5

1. The basics of DNA replication

2. Replication initiation, progression and termination

3. Unique aspects of eukaryotic DNA replication

Outline

High-fidelity for genome integrity and survival

Human genome : 3G bp

Chemistry of DNA synthesis

1. Template + Primer

2. Base-pairing

3. 5’->3’ polymerization

(dNTPs)

Primase generates RNA primer

** No de novo DNA synthesis by DNA polymerase

DNA Polymerase

** 5’->3’ ** No de novo synthesis

Positioning-> conformational change-> polymerization-> pyrophosphate release ->Conformational change->translocation of the DNA for 2nd reaction.

Bidirectional DNA Synthesis

Autoradiograph of an E. coli chromosome in the act of replication

Parental DNA

Newly synthesized DNA

Partental

Daughter

Daughter

Figure 5-7 Molecular Biology of the Cell (© Garland Science 2008)

Okazaki fragments: ~1000 nt (Bacteria)~100-200 nt(Eukaryotes)

Asymmetrical Replication

Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)

Lagging strand synthesis

Initiation, Progression, Termination

oriC: origin of Chromosomal replication

Bacterial origin of replication

-DnaA box TTATCCACA

-AT-rich region

-GATC (13)

DnaA binding

DnaA binding

DnaA binding

Dam Methylation Controls Origin Firing

!!! Plasmids from bacteria are Dam methylated !!!

Plus of 3H-thymidine

Multiple Origins of Eukaryotic Replication

BacteriaCircular, naked DNASize: 4*106

Replication: ~600-730 nt/s

EukaryotesLinear Chromatin: DNA+ nucleosomeSize:3*109 (Human)Replication: ~17-33 nt/s

Budding yeast 400 Ori, 100-200 bp:A domain- 11bp ARS consensus sequence (A/TTTTATG/ATTTA/T) for ORC bindingB domain-AT richStrong v.s. weak origins

Yeast ARS: Autonomously replicating sequence

Vertebrate Origins

• AT rich DNA with no strong sequence preference.

• Zones of initiation span 1-100 kb at different loci (i.e. DHFR, LMNB2, IGH…).

• Controlled by epigenetic states, DNA topology, transcription.

• Developmental and cell cycle regulated

BrdU plus-labeling ~ 1 Mb replication foci

Distinct replication timing of different chromosomal regions

3D Genome

Gonzalez-Sandoval A & Gasser SM, 2016, Trens in GeneticsMatharu N & Ahituv N, 2015 PLOS Genetics

Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization. Miura et al., Nature genetics, 2019

!!! S phase and only once !!!

Late M/G1

Early S

S

Initiation ORC complex

Cdc6 + Cdt1+ MCM (inactive)

MCM + Initiation factors(Cdc45, GINS..)

MCM + RPA + polymerase + PCNA…Initiation factors

Prereplication complex

Cdc6/Cdt1 displacedInitiation factors recruitmentMCM activation upon Phosp

Replication factorsrecruitment

MCM Helicase loading

Preventing pre-RC formation in S/G2/M

Geminin inhibits Cdt1

1. CDK/DDK phosphorylate Cdc6 and Cdt1 in S phase.

2. Geminin inhibits Cdt1 in S/G2/M and is degraded in G1

Replication Progression

!!! Co-directional movement of leading and lagging polymerases with helicase !!!

Active replication fork

T4 phage replication machine

Replisome: Primase, Polymerases, Helicase, PCNA, Clamp loader, RPA, Topoisomerase……..

DNA helicase (MCM)

MCM: minichromosome maintenance

Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008)

SSB (RPA) straightens ssDNA template

Non-specific, fast on and off

Sliding Clamp (PCNA): Processivity factor

PCNA (Proliferating cell nuclear antigen): Keep polymerase on the DNA

Replication Factor C (RFC)

600 nucleotides / second

DNA Tangling During Replication

Type I Topoisomerase

Figure 5-23 Molecular Biology of the Cell (© Garland Science 2008)

Type II Topoisomerase

TopII Separates Daughter Chromosomes

TopII

Eukaryotic Replication Termination

Unique Aspects of Eukaryotic DNA Replication

1. Nucleosome assembly

2. The end replication

Replication-coupled nucleosome assembly

Histone octamer:

2*H2A-H2B dimer +

H3-H4 tetramer

1. Deposit of H3-H4 tetramer2. Deposit of two H2A-H2B

Nucleosome re-assembly is coupled with DNA synthesis-CAF-1-PCNA, Asf1-MCM, Asf1-RFC, FACT-polymerase

(H3-H4)2 Chaperone: Asf1, CAF-1, Rtt106(H2A-H2B) Chaperone: FACT, NAP1

NSMB 20, 4-22, 2013

Histone Chaperones mediate nucleosome re-assembly

The end replication problem

DNA polymerases possess two unusual features:

1. They synthesize DNA only in the 5’ to 3’ direction

2. They cannot initiate DNA synthesis

Telomerase for Telomere Elongation

In humans, telomerase is expresses in germ cells, but not in somatic cells.

TetryhymenaEuplotesOxytrichaS. cerveisiaeS. PombeVertebrate

TTGGGGTTTTGGGGTTTTGGGGT(G)2-3(TG)1-6G2-8TTAC(A)TTAGGG

~400bp 28 bp20 bp~300 bp~300 bp5-50 kbp

Telomeres

Eukaryotic replication proteins

Helicase loading proteins/Licensing factors

Initiation factor (pola-pirmase recruitment )

MCM Helicase

Primase & DNA pol

PCNA

Clamp loader RFC complex

TOPI&II

RNase

Ligase

RPA

ORC complex