Eukaryotic DNA Replication

1) Comparison with bacterial DNA replication 2)Reverse transcription 3)The end replication problem
Telomeres and telomerase
Telomeres , the physical ends of chromosomes

Composed of --G-rich repeat (10-1000s) with ss overhang --Associated proteins

The Eukaryotic Replisome

The eukaryotic replisome is homologous in many respects to the bacterial replisome!!!
Pol - the eukaryotic replicase Pol /primase - contains both primase and DNA polymerase activities PCNA - trimeric sliding clamp Replication Factor C (RFC) - the clamp loader MCMs - a heterohexameric helicase Replication Protein A (RPA) = SSB RNase H - nuclease that is specific for RNA in RNA/DNA hybrids excises primers

The Eukaryotic Cell Cycle

Each chromosome has multiple origins. Why? Time for DNA replication is limited - e.g., mammalian S phase lasts 6-8 hours. Eukaryotic forks move at only about 1/10th the rate of bacterial forks Chromosomes can be in excess of 108 bp Completion of replication in the allotted time necessitates multiple origins.

Origin Recognition Complex (ORC) - a complex of 6 ATPases - the functional equivalent of DnaA

Reverse Transcription
How do retroviruses replicate? RNA genome must be copied to DNA for a) integration into host genome b) synthesis of viral mRNA and genomic RNA Enzyme: reverse transcriptase (RT) an RNA directed, DNA polymerase uses RNA template to make DNA! RT activities a) 53 RNA directed DNA pol (synthesizes DNA on RNA template) b) 53 DNA directed DNA pol (synthesizes DNA on DNA template) c) exoribonuclease (RNase H) (degrades RNA in RNA:DNA hybrid) During infection,
dNTP RNA (1970 Temin & Baltimore labs)

RT transcribes RNA to RNA:DNA hybrid (a) RNase H activity degrades the RNA (c)
DNA

NMPs

RT makes ds DNA from ss template (b) ds DNA directs rest of infection

The End Replication Problem

Fork movement

origin
Fork movement
lead ing

5 3

lag gin g
lead ing

3 5

lag gin g

5
RNaseH

3
RNaseH

The solution is provided by a special reverse transcriptase called telomerase!!

Telomeres , the physical ends of chromosomes

Telomeres & Telomerase

Telomere: physical end of chromosome. Usu composed of ds DNA with 101000s of G-rich repeats (on lagging strand) and associated proteins
Composed of --G-rich repeat (10-1000s) with ss overhang --Associated proteins

DNA is ds (G rich in one strand), except at 3 end where at least 2 repeats form a ss 3 overhang. e.g. human TTAGGG Oxytricha nova TTTTGGGG Tetrahymena TTGGGG 5 3 (TTTTGGGG)n TTTTGGGGTTTTGGGG (AAAACCCC)n

The end of the G-rich strand is synthesized by telomerase a riboprotein! -- contains RNA & protein
(159 nt in Tetrahymena)

(several subunits)

Protein that catalyzes nt addn has homology to RT!

Replication of telomere ends by telomerase:

A riboprotein composed of RNA (hTR) which includes an RNA template and proteins which include the catalytic reverse transcription (hTERT).
5 3

GGTTAGGGTTAGGGTTAG3
5

elongation
5 3

TTGGGGTTGTTAGGGTTAGGGTTAG CAAUCCCAAUC

telomerase RNA

) )

template

translocation of telomerase (with template RNA)

5 3

etc.

TTGGGGTTGTTAGGGTTAGGGTTAG GGTTAG CAAUCCCAAUC CAAUCCCAAUC template telomerase telomerase 5 5 3 3 RNA RNA

The other (leading) strand is replicated by the normal DNA replication machinery.

Secondary structure of human telomerase RNA determined by phylogenetic comparison of 32 vertebrates hTRs

Telomerase: a large RNA and protein complex

Known protein interactions with human telomerase RNA

CR4/CR5

catalysis

CR7

Nop10

hTERT
Pseudoknot H/ACA

Nhp2

dyskerin Gar1

Template

localization

The highly conserved pseudoknot sequence in the pseudoknot (core) domain is essential for enzyme function

Mutations linked to dyskeratosis congenita and aplastic anemia

Structure of the conserved pseudoknot in human telomerase RNA

The U-rich loop 1 lies in the major groove of stem 2 and the A-rich loop 2 lies in the minor groove of stem 1, both on one face of the pseudoknot
Three stem 2-loop 1 major groove triples and two stem 1-loop 2 minor groove triples flank the helical junction to form a continuous triple helix

Stem 1 Loop 2

Loop 1

Stem 2

Theimer, Blois, Feigon, Mol. Cell (2005)