9. Dna Replication and Telomeres

DNA Polymerase responsible for synthesis of new DNA strands

enzyme from e.coli capable of in vitro dna synthesis

polymerase I

single polypeptide of 109,000 daltons

Dna POl I mutants

some viable and made DNA at normal rates, not major enzyme dna replication in vivo

dna pol III

large complex protein composed of 10 diff polypeptides, key enzyme synthesis of DNA from RNA primers Dn

dna polymerase polymerization

addition of nucleotide triphosphate, two phosphates are removed and p1 is bonded to 3’ OH end of primer

DNA core polymerase

alpha, epsilon, theta

• alpha active site for nucleotide addtion in 5’ - 3; direction

• epsilon 3’-5’ exonuclease (activity that proof-reads)

b subunit of pol III

functions as a clamp, it associates with core at 3’ end of the growing strand, increases the processivity of polymerase

gamma complex of pol III

loads and unloads sliding clamp onto dna template (atp required), includes Tau protein (2 copies) allows dimerization of 2 core polymerases so leading and lagging synthesis are coordinated

3’-5’ exonucleolyitc proofreading contributes to high fidelity of DNA synthesis

if new base is incorrect, it is hydrolyzed and expelled

RNase H

removes most of RNA primers’ nucleotides ( except the last one) , auxiliary role, mostly if longer primers

Dna Pol I

can remove ribonucleotides, (including “last”), fills gap with deoxyribonucleotides (5’-3’ normal polymerization activity) not highly processive

Dna Pol I 5’ - 3-

removes ribonucleotides and 3’ - 5’ proofreading

Dna Ligase

links dna fragements on the lagging strand following primer removal and Pol I synthesis by creating phosphodiester bond between the 3’ - OH and the 5’ phosphate of adjacent nucleotides. ATP Required

two types of topoisomerase

I and II (gyrase requires atp)

topo introduces negative supercoils around OriC

necessary for initiation of replication by DNaA

after helicase’s action positive supercoils are formed ahead of a growing fork

topo II converts them into negative supercoils

leading strand core elongates leading strand from

RNA primer in direction of growing fork

lagging strand core synthesizes

okazaki fragment from RNA primer (loop is formed ebtween lagging core and growing fork)

primase is positioned by helicase so it could

make a new rna primer (10-15nt) for the lagging strand

loop is growing; lagging core

displaces SSB and finishes Okazaki Fragment

completion fo replicaiton of circular DNA

1. last few helical turns in the parental DNA have to be removed

2. nearly completed daughter helices create cateanes

3. denaturation of unreplicated terminus followed by supercoiling

4. replication proceeds to completion before or after decatenation

Topoisomerase IV (belongs to topo II fam)

responsible for decatenation in vivo

Topoisomerase II

catalyzes decatenation of interlocked catenanes in vitro but cannot substitue for topoisomerase IV in vivo

eukaryotic replication complexity

1. complex chromatin structure

2. multiple replicons

3. replicons 40-100kb long

4. euchromatin first

5. domino model of origin activation progression during synthesis

Autonomous replication sequence elements in S.cerevisiae

1. ars is OriC in yeast

2. 11bp sequnce called A-domain is conserved

3. mutation in A-domain of ARS aboligins origin function

4. B1, B2, B3 are nearby elements; not conserved

5. mutations in these elemnts reduce but do not prevent origin function

Eukaryotic Origin Recognition Complex ORC

1. ORC dna replication initiator protein

2. its binding identifies an origin of replication

3. remains associated with ARS element through cell cycle

4. initiation must depend on binding of additional proteins or on change in ORC (not on formation of a new association)

CDC 6 cell division cycle kinase 6

• unstable t1/2 < 5 min

• synthesized, binds to ORC during G1

• has ATPase needed for initiation

• binding allows other proteins to bind

Yeast MCM 2-7 mini chromosome maintenance

• bind to orc complex if CDC6 bound

  • needed for initiation

cdt1 (cdc10-dependant transcript 1)

• part of licensing factor

• accumulates from M to G1, degrades at S

licensing factor (protein) Gdt 1- geminin regulates levels

• inactivated or destroyed in the nucleus after replication round

• new replication round requres new licensing factor to enter nucleus (translated in the cytoplasm)

• re-entry believed to occur in subsequent mitosis when nuclear membrane breaks down

Pol alpha eukaryotic dna pol

priming dna synthesis during replication and repair

pol delta eukaryotic dna pol

dna replication of lagging strand during replication and repair

pol epsilon of eukaryotic dna pol

dna replication of leading strand during replication and repair

FEn-1

endo/exonuclease - removes primer from both strands (sometimes need RNase H)

• coordinated by PCNA

chromosomes lose about 100 base pairs from their 5’ ends in each mitosis

genetic info is getting lost with each division

telomere

• dna sequence consists of a simple repeating unit (6-7 nt) with a protruded single-stranded 3’ end that may fold into a loop)

human telomere sequence

TTAGGG - G-rich strand

telomerase

enzyme that repairs and replicates the ends of chromosomes

ribonucleoprotein

protein-rna complex

telomerase carries its own little template rna and has

reverse transcriptase activity (dna from rna template)

telomerase adds nucleotides to 3’ OH end of the lagging strand template to extend it

polymerizes dna accrding to its own rna template

after elongation of 3’ end of the lagging strand template,

new rna primer for the lagging strand will be made by primase and polymerization will continue by polymerase, new rna will be degraded, resulting in an extra 3’ overhang of the lagging strand template

yeasts, unicellular eu organisms, cells that proliferate indefinitely

possess homeostatic mechanisms to maintain telomere length within a limited range

telomere length increases early in development

somatic cells are “born” with full complement of telomeric repeats

some human cells do briefly express telomerase during S phase but is

turned off in some tissue (skin) length maintained by DNA recombinations in telomeres

telomerase generally found only in

1. unicellular eukaryotes

2. germ cells, incl es cells

3. cancer cells

aging cells e.g skin do replicate in

absence of telomerase activity

replicaative cell senescence

descendent cells will inherit defective chromosomes, such cells will eventually withdraw permanently from cell cycle and stop dividing

shelterin

mammalian telomeres consist of tandem repeats of the TTAGGG sequence that are bound by the shelterin-telosome protein complex. adjacent to telomeres are the subtelomeric regions, which are also rich in repetitive DNA

methylstion

In addition to Shelterin, mammalian telomeres also contain nucleosomes that show histone modifications characteristic of heterochromatin domains.
In addition, subtelomeric DNA is heavily methylated. These chromatin modifications at telomeres and subtelomeres have been shown to negatively regulate telomere length and telomere recombination. (TriM = trimethyl.)