The document discusses the concept and process of DNA replication. It begins by defining replication as the copying of genetic information that is inherited to subsequent generations. It then summarizes the three proposed models of replication before discussing Matthew Meselson and Franklin Stahl's experiments, which supported the semiconservative model. The rest of the document details the specific mechanisms and steps of prokaryotic and eukaryotic replication, including initiation, elongation, termination, origins of replication, and the roles of various proteins. It concludes by discussing the importance of telomeres and telomerase in preventing chromosomal shortening during replication.

1. CONCEPT
OF
REPLICATION
By,
Ashwin Shivashankar
Athira Mary Rajan
Chandana B L
Chinchu Ouseph
Deepak A P

2.  Copying of genetic information
 Inherited to next generation or to the
daughter cells of cell division
 Allows to receive complete copy of
genetic information
 Prior to cell division during the S phase of
interphase

3.  At the end of their 1953 paper Watson
and Crick wrote-
“It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible copying
mechanism for the genetic material”
 As two strands are complementary, one
could act as template and determine the
sequence of other

4.  After Watson and Crick proposed the double
helix model, 3 models for DNA replication were
proposed
1. CONSERVATIVEMODEL
- two parental DNA strands binds back after
replication
- one daughter molecule with both parental
strands and the other with two newly
synthesized strands

5. 2. SEMICONSERVATIVEMODEL
-the daughter molecules will have one parental
strand and one newly synthesized strand
3. Dispersivemodel
-daughter molecules each with parental and
progeny DNA segments interspersed

6.  Matthew Meselson and Franklin Stahl ( 1958 )
 Controlled isotopic composition of nucleotides
incorporated in the newly synthesized daughter
DNA
 E.coli grown for several generations in media in
which all nitrogen were normal isotope 14N;
culture served as control
 Other culture with E.coli grown for several
generations in media with only N source was
heavy isotope 15N

7.  Culture with E.coli having 15N labeled DNA
transferred to new medium were all nitrogen were
14N
 Any DNA synthesized after transfer would contain
lighter isotope
 Isolated DNA from cells grown in different N-
isotope cultures and subjected to equilibrium
density gradient centrifugation
 15N DNA denser than 14N DNA thus forming band
closer to bottom of tube
 Resultant 1st generation cells formed band at
density intermediate to pure 15N DNA and pure 14N
DNA

9.  Specific base pairing
 Begins at sequences called origins
 Initiated by short fragments of RNA called
primers
 Elongation of DNA strand always in 5‘-3‘ direction
 New nucleotides added to 3‘-OH group of
growing strand
 New nucleotide strands complementary and
antiparallel to template strand

10.  ORIGIN : where replication begins
 HELICASES : unwinding proteins of double helix
 SINGLE-STRANDED BINDING PROTEINS : stabilizes
single stranded regions of DNA
 TOPOISOMERASES : relaxes stress further up the
strand
 DNA POYMERASE : 5‘-3‘ DNA synthesis
 PRIMER : a segment of new strand with 3‘ hydroxyl
group to which nucleotides can be added
 PRIMASE : synthesize RNA primers
 LIGASE : fills in final gaps on lagging strand

11.  3 major steps –
1. INITIATION
2. ELONGATION
3. TERMINATION

12.  Occurs in 5‘-3‘ direction at a rate of 1000
nucleotides per second
 3 types of polymerases
- DNA pol I – exonuclease activity
- DNA pol II - repair function
- DNA pol III – DNA synthesis
 Single origin of replication

13.  Origin of Replication
 245 base pair long
 AT rich sites
 9 bp and 13 bp repeats
-DnaA box (4 nos.): 5’ - TTATCCACA - 3’
-DnaB box (3 nos.): 5’ - GATCTNTTNTTTT - 3

14.  INITIATION
 DNA binds around initiator protein complex DNA-A
 DNA-B or helicase unwinds ori C
 SSB binds to single stranded region to protect it
from breakage and prevent premature reannealing
 DNA gyrase relives stress ahead of replication fork
 DNA primer attaches to DNA and synthesis a short
RNA primer

16.  ELONGATION
 DNA pol III is loaded and it extends the RNA primer
 Leading strand :
- the new strand continuously synthesized in
5‘-3‘ direction in the 3‘-5‘ oriented template strand
 Lagging strand :
- the new strand discontinuously synthesized in
small fragments in 5‘-3‘ oriented template strand

17.  After synthesis by DNA pol III, with its 5‘-3‘
exonuclease activity DNA pol I removes RNA primer
and fills gap with DNA
 DNA ligase fills gaps and nicks

18.  Termination
 Two replication forks meet
 Terminator sites arrest the movement of forks by
binding to tus gene products (inhibitor of helicase)
 Two interlinked double stranded DNA molecules
(catenanes) separated by topoisomerase II

20.  Multiple origin of replication
 Formed of about 150 nucleotides
 Replication occurs at various points simultaneously
 Slow, at a rate of 100 nucleotides per second

21.  PRE REPLICATIONCOMPLEX
 Protein complex that forms at the ori
 Assembling of complex occurs during late M phase
and early G1 phase when CDK activity is low
 This timing and other regulatory mechanisms
ensure that DNA replication occur only once during
the cell cycle
 Complex activated during S phase
 Separation of assembly and activation to different
phase prevents origin firing more than once in a
single cell cycle

22.  Termination
 DNA pol halts on reaching a section of DNA
template that has already been replicated
 DNA pol cannot catalyze phosphodiester bond
formation between two segments of new DNA
strand and it falls off
 These unattached sections of full replicated DNA
strands are called nicks
 Enzymes FEN1 and Rnase H remove RNA primers

23.  Once RNA primers are removed free floating
DNA pol lands at 3‘ end of preceding DNA
fragment and extends DNA over gap
 Eukaryotes lack termination sequences and
proteins analogous to Ter sites and Tus protein
in prokaryotes

25.  Protective caps at ends crucial in preserving the
structural integrity of each chromosome
 DNA pol can reconstruct only the 3‘ end and not the
5 end
 If left to its own devices, chromosomes of
successive generations of cells would become
shorter and shorter
 Telomeres and an enzyme telomerase
countermeasure to this problem

26.  Special repetitive sequence of telomeres
contain no protein coding genes
 Telomerase - carries its own RNA template with
it
 Telomerase also has several additional protein
domains needed to assemble the enzyme at the
ends of chromosomes properly

28.  When eukaryotic DNA is replicated, it
complexes with histones.
– This requires synthesis of histone
proteins and assembly of new nucleosomes.
 Transcription of histone genes is initiated
near the end of G1 phase, and translation of
histone proteins occurs throughout S phase.