01:694:301 Lecture Notes - Lecture 28: Okazaki Fragments, Dna Polymerase I, Dna Replication

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Chapter 28‘DNA Replication, Repair, and Recombination’
The central concept of this chapter is the replication of DNA and the focus of replication is
the Replication Fork (the site of DNA synthesis) (Fig 28.9, 28.21)
o A double helix separated into two single strands can be replicated because each strand
serves as a
template
on which its complementary strand can be assembled
o Replication has to be extremely accurate; this is achieved through multilayered of DNA
synthesis ! proofreading, post replication mismatch repair
o DNA synthesis always proceeds 5’ ! 3’ direction; special mechanism is
used to replicate antiparallel strands
o Strands must be separated to generate appropriate template; helix needs
to be unwind
DNA polymerases require a template and
primer
o DNA Poly catalyzes the formation of polynucleotide chains; ‘template
directed enzymes’
" Incoming dNTP form the ‘right’ base pair with base in template
" DNA polymerases require a primer with a free 3’—OH group already
base-paired to the template; nucleotides are added to 3’ end
" DNA poly close down incoming dNTP that are bound to poly active
site; conformational change forms tight pocket where
the right base pair can fit readily
o
Primase
, an RNA poly, synthesizes short stretches of RNA
complimentary to DNA
" After DNA synthesis begins, RNA segments removed by hydrolysis
Understand how Okazaki fragments are formed and changed into DNA on the lagging strand
o
A significant proportion of newly synthesized DNA exists as these small fragments
o These fragments are joined by
DNA ligase
to form continuous daughter strand
o The strand formed from okazaki fragments ! lagging strand; leading strand is
synthesized w/o interruption
o ‘Nick translation’ - DNA Pol I replaces RNA primers of okazaki fragments with DNA; gaps
remaining needs to be sealed
o DNA ligase
seals the breaks (gaps) in double stranded DNA molecules
(It does NOT join 2
single stranded DNA)
The separation of DNA strands requires helicases and ATP hydrolysis
o For replication to occur, DNA strands are separated allowing them
to act as templates
o Specific enzymes,
helicases
utilize ATP hydrolysis to power strand
separation
o Helicases contain six subunits that form a ring structure; each
subunit has P-loop NTPase
o *Note that helicase is homologous with F1 ATPase in mito (and rho protein)
As helicase unwinds DNA, torsional stress can cause
supercoiling
o Supercoiled DNA is more compact than relaxed DNA
o Linking number is a topological property of circular DNA; it is equal # times
strand of DNA winds in right handed direction around helix axis
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