DNA Replication

Overview of DNA replication

• occurs in S phase of interphase in nucleus for eukaryotic organisms

• Semi-conservative process

  • Parents strands of DNA separate

  • Each serves as template synthesis of new complementing daughter

  • Product = DNA contains 1 old and 1 new strand

Steps of replication

Initiation, Synthesis/Elongation, Termination, DNA Proofreading Function

Initiation

occurs @ origin = specific DNA seq. (-250 BP)

• 1 origin in prokaryotes

• many origins on each linear euk. chromosome

Origination Factors

• DNA binding proteins recognize + bind to origin

• causing strand of helix to separate

Single strand DNA binding proteins

SSBs bind to SSDNA and prevent internal folding

Replication bubble

where the DNA strands separate

Replication form

where SSDNA and DSDNA meet

Helicase

enzyme binds replication forks + opens/unzips helix

Supercoils

form downstream as the helix unzips and opens

Topoisomerase

alleviates super coils

Synthesis/Elongation

Each parent strand serves as a template to make new complementary DNA strand 5’ - 3’

Primase

(special RNA polymerase) creates short (4-6 nts.) RNA primer 5’ - 3’

DNA Polymerase 3

adds DNA nts. starting at primer

• polymerase enzymes (pol) → synthesize nt strands 5’-3’

• Rxn: dNTP + 3’ OH of growing nt strand

  • deoxynucleotide triphosphate

  • strand + 1 nt. + PPi (pyrrophosphate)

Continues strand

strand replicated towards the fork is replicated continuously as fork opens until termination

Discontinuous/lagging strand

Synthesis of other strand is said to be discontinuous/lagging strand synthesis

• Now RNA primers added (-150 nt.) as the fork opens

• DNA Pol. 3 extends primer adding nts. 5’-3' until next primer reaches

• DNA Pol. 1 Removes RNA nts using 5’-3’ exonuclease activity

• DNA Ligase seals gap (forming phosphodiester bond) between strand

Prokaryotic replication termination

2 new strands

Eukaryotic replication termination

• leading strand replication occurs until next strand met (ligase seals) or the end of chromosome. DNA Pol. synthesized to end + falls off

• lagging strand synthesis, end of chromosome cannot be completely replicated + nucleuses

  • Problem = ultimately less genetic info

  • Sol’n = Telomere - DNA seq (-7 BP) repeats 100’s of times @ end of linear

    • Telomere’s synthesized by telomerase

Telomerase

• enzyme that creates telomerase comprised protein/enz. (polymerase) + SSRNA template

  • “on” (expressed/present) in developing cells + stem cells (unlimited division potential)

  • “off” in adult differentiated specialized

    • telomeres shorten w/ each cell division → act as biological cloth

    • shorter telomere → more divisions have occurred

    • too short → cells kills itself = apoptosis

DNA Proofreading Function

corrects errors of incorporation

• DNA Pol. 3 back up once error is detected 3’-5’ temporarily and utilizes 3’-5- exonuclease function to remove error