GBS 708 - Week 2

ORC

• position on DNA at which replication start points are found by physical methods (where do nascent strands of DNA start?)

• DNA sequence that when added to non-replicating DNA, it causes it to replicate.

• DNA sequence whose mutation abolishes replication

• 6 subunit protein complex (ORC1-ORC6), binds to A box of ARS1 and replication origins in an ATP-dependent manner; ATPase activity

• is required to load (CDC6, Cdt1, MCM2-7)

Autonomously Replicating Sequences (ARS) + image!

in yeast, if adding DNA fragment to non-replicating DNA causes it to replicate, then there is an ORC on fragment

ARS1

• origin in plasmid, point mutations (with URA3 gene as selection marker) identifies critical A elements in ARS1

• WT-ARS1: transform ura3-yeast with URA3 plasmid (with different mutations across ARS) and plate on uracil

SV40

• polyomavirus, if you delete origin, virus does not replicate

• double-stranded DNA circular virus that replicates like plasmid in mammalian cells

7 replication elongation factors/machinery (proks vs euks + function)

are conserved!

8 steps of DNA replication

1. T-antigen/Helicase binds origin on DNA and unwinds DNA. Replication-Protein-A (RPA) binds single-stranded DNA

2. RPA coats single-stranded DNA and recruits DNA pol α primase

3. DNA pol α primase synthesizes RNA primer (2-6 bases) that copies DNA

4. DNA pol α extends RNA primer as DNA only in 5’-3’ direction; it lacks proof-reading activity and so DNA is error-filled

5. RF-C loads PCNA at DNA end, which displaces DNA pol α and recruits/attaches DNA pol Δ/ε to DNA which synthesizes rest of new leading strand/Okazaki fragment

6. DNA pol α and DNA pol α primase initiates new Okazaki fragments on lagging strand. Pol switching occurs on lagging strands; pol δ/ε (have proof-reading activity) do most of DNA synthesis

7. RNAseH, 5→3 exonuc Fen1, and Dna2 endonuc remove RNA primers/RNA and most of DNA pol α synthesized-DNA (bc error-filled) at 5’ end of each Okazaki fragment

8. DNA ligase I uses energy of ATP hydrolysis to seal nicks in DNA

RF-C

• 5-subunit complex; all subunits related in sequence and have ATP binding motifs

• hydrolyze ATP to load PCNA clamp

• functional homolog of clamp-loader γ complex

3 steps of SV40 DNA replication in vitro experiment

1. COS cells (infected with SV40 and expresses TAg) + cell lysate + plasmid w/ ori + dNTP + rNTP + α32P dATP @ 37C for 60 mins

2. DNA replication will incorporate radioactive α32P dATP into 5 kb DNA → analyze products by electrophoresis and autoradiography

3. fractionation of cell lysate to homogeneity to identify replication factors provided by cell

proof-reading activity

• 3→5 exonuc activity in DNA pol δ and ε, NOT IN DNA pol α!!!

• primase in 2 separate subunits of DNA pol α lays down primer

PCNA clamp vs β clamp (compare vs contrast)

• PCNA in euks, β clamp in bacteria

• functionally same and structurally similar, forms protein ring around DNA

• clamp pol to DNA, processivity factors for DNA pols

Processivity

can pol stay attached to its product DNA and extend it for long stretches?

DNA Helicase

• hydrolyzes ATP to move/binds ss DNA and peel annealed DNA and unwind double-stranded DNA

• moves 5→3 or 3→5 (so both directions) on ssDNA; conserved, dimer or hexamer

• binds ATP → tight conformation; hydrolyzes ATP → relaxed conformation

• Walker A motif (GxGxGKT) and Walker B motif (ΦΦΦDEad) for ATP-binding/hydrolysis; these are also present on all proteins that hydrolyze ATP (helicases, clamp-loaders, protein kinases, DNA ligases, ATPases)

MCM2-7 (Mini-Chromosome Maintenance) + image

• core of euk replicative DNA helicase; if yeast has temp-sensitive (ts) mutation in replication factor, then at nonpermissive temp, plasmid will not replicate and will get lost from culture

• yeast containing Leu2 on plasmid → grow under non-selective conditions (permissive/nonpermissive temps) → plate yeast on Leucine-plates to estimate rate of plasmid loss

• inactive, consists of hexamer of MCM #2-7, loaded on ds DNA in G1 using cdk2 kinase @G1-S→ creates CMG complex (active, consists of MCM2-7, cdc45 and GINS), an active helicase poised for DNA unwinding

• nuclear export (in yeast) or displacement from chromatin (in mammalian cells).

3 initiator proteins

Mcm2-7 helicase core cannot load on DNA by itself and requires help of initiator proteins (ORC, Cdc6, Cdt1)

CDC6 and Cdt1

• essential factor that cooperates with Cdt1 (degradation in mammalian cell) for loading of MCM2-7

• proteolyzed in yeasts or exported out of nucleus in mammalian cells at G1- S transition

• overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, critical regulator of DNA replication

• DNA replication initiation required on double-stranded DNA substrate is dependent on Cdc6, elongation not effected!

• overexpression of Cdt1 and Cdc6 produces cells with > 4n DNA

cyclin-CDK complex + know image

• dimeric enzyme that is active/inactive as cell moves through cell-cycle

• consists of cdk (catalytic subunit) and cyclin (regulatory subunit)

• cdks phosphorylate substrates on Serine (S) or Threonine (T)

• different cyclin-cdk complexes drive different parts of cell cycle

• initiation of replication at G1-S transition

• prevention of re-replication before mitosis AND mitosis before replication

Initiation of replication at G1-S transition

• CDK converts inactive MCM2-7 to active CMG helicase

• CDC45, GINS, CDC7/DBF4, CyclinE/A-Cdk2

cdc45

• activates MCMs along with GINS (CMG complex) to initiate DNA replication

• unwinds DNA, loading of DNA primase/polymerase complex

GINS (Go-Ichi-Ni-San complex)

• essential in initiation of DNA replication, activates MCM2-7 helicase

• 4 subunits (SLD5, PSF1, PSF2 and PSF3) that forms ring on EM.

CDC7/DBF4 + know image!

• DBF4-dependent-kinase (DDK) and Dbf4 required for initiation of DNA replication

• kinase phosphorylates MCMs to relieve auto-inhibition

CyclinE/A-Cdk2 kinase + know image!

• protein kinase essential for initiation of DNA replication by phosphorylation/activation of Sld2 + Sld3 substrates to recruit Dpb11 adaptor, CDC45 and GINS-polε to form active CMG

• assembles CMG w/ DDK (de-represses MCM), essential for activating MCM2-7 helicase to form active CMG

unwinding to DNA synthesis: recruitment of which 2 polymerase-primases?

• Mcm10 and AND-1 (and pol ε) recruit DNA pol α to CMG helicase and onto ssDNA

• MCM10: essential in initiation of DNA replication in yeast, required in Xenopus after loading of Mcm2-7 and before loading of CDC45, splits Mcm2-7 double hexamers into 2 hexamers

• AND1/CTF4: in yeast, mutations lead to defects in chromosome-transmission-fidelity. required for stability of p180 (DNA pol α, catalytic subunit) and to load pol α on chromatin at ORC along with polymerase ε

2 ways cell prevent re-replication in the same cycle?

• cdks phosphorylate replication initiator to prevent loading of new MCM2-7 on replicated DNA

• geminin stabilized in S phase to also prevent loading of new MCM2-7

Geminin

• in Xenopus, protein degraded by Anaphase-Promoting-Complex

• inhibits initiation of DNA replication by blocking loading of MCM proteins onto replication origins

• inhibits Cdt1 in S phase to prevent re-replication, knockdown causes more RNAi

5 steps in checkpoint pathways

cyclin-cdk prevented from becoming highly active before DNA replication complete so cells cannot enter mitosis while DNA replication ongoing

1. ATR/ATM kinases activated when DNA replication ongoing (re-replication)

2. geminin loss causes re-replication that activates Chk1 and Chk2, Chk1/Chk2 kinases phosphorylated

3. Cdc25C phosphatase phosphorylated

4. 14-3-3σ binds to phospho-Cdc25C and sequesters/inhibits it in cyto

5. Cdk1 (Y15-P) inactive

4 anti-S phase (de-regulate DNA replication) anti-cancer drugs

• topoisomerase inhibitors (etoposide, doxorubicin)

• alkylating agents

• cross-linking agents (cisplatin)

• radiation

activate intra-S phase checkpoint and block cell-cycle! induction of re-replication kills cancer cells