Post on 27-Feb-2020
11/19/2014
1
19.11.2014 1
What is replication?
Unique property of double stranded molecules of DNA
Semi-conservative synthesis of DNA based on a template
Duplication of genetic material
The main mechanism that assures transmission of information from cell to cell
and from one generation – to another.
19.11.2014 2
Why replication is required?
Assures exact transmission of
information
Determines self-
reproduction
Represents the support of heredity
Assures material linkages between
generation
19.11.2014 3
11/19/2014
2
When replication takes place?
19.11.2014 4
What are the principles of replication?
Based on template;
Complementary;
Antiparallel;
Two directions;
Semi-conservative;
Fidel;
Very complex.
19.11.2014 5
19.11.2014 6
11/19/2014
3
Bidirectional synthesis
19.11.2014 7
Principles of replication:
The monomers (nucleotides) are added to 3’ of one strand by enzyme DNA
polymerase;
Newly syntheses strand is complementary to template strand;
Synthesis is semi-conservative: each new double-stranded molecule
contains an old strand (template strand) a new strand (daughter strand).
19.11.2014 8
Semi-
conservative
mechanism
19.11.2014 9
11/19/2014
4
19.11.2014 10
19.11.2014 11
Components required for replications
DNA molecule - template
Origin of replication – point ORI
Enzymes
Nucleotides (dNTP and NTP)
SSB proteins
19.11.2014 12
11/19/2014
5
19.11.2014 13
Main enzymes required for
replication DNA-polymerase (I, II,
III – in prokaryotes, a, g, e – in eukaryotes)
Primase
DNA-helicases
Topoisomerase
DNA-ligase
Telomerase
19.11.2014 14
DNA
Polymerase
19.11.2014 15
11/19/2014
6
Characteristics of eukaryotic
DNA-polymerases
DNA polymerase α δ ε β γ
Location Nucleus Nucleus Nucleus Nucleus Mitochondria
Synthetic activityPrimer
extension
Synthesis of
DNADNA repair DNA repair Replication
Additional
functions- Exonuclease Exonuclease - Exonuclease
19.11.2014 16
19.11.2014 17
Topoisomerase
19.11.2014 18
11/19/2014
7
Which are the main steps of
replication?
Initiation
Elongation
Termination
19.11.2014 19
Initiation of replication:
Initiation of polymerization of DNA
Synthesis of RNA primers
Formation of replication eye containing two replication forks:
Unfolding of DNA Denaturation of DNA
Identification of point ori;
19.11.2014 20
Point of initiation of replication - ORI
• consists of ~300 bp
• contains sites for interaction with specific proteins required for initiation of replication
19.11.2014 21
11/19/2014
8
19.11.2014 22
Elongation
Enlarging of replication eye;
Synthesis of DNA in two directions:
Continuous synthesis of leader strand;
Interrupted synthesis of lagging strand; synthesis of Okazaki fragments on lagging strand.
19.11.2014 23
19.11.2014 24
11/19/2014
9
19.11.2014 25
19.11.2014 26
Termination
1 mol. DNA 2 mol. DNA.
Renaturation of DNA;
Ligation of all fragments;
Feeling in the gaps;
RNA primers are removed;
Meeting of opposite replication forks;
19.11.2014 27
11/19/2014
10
The replicon – replication unit
Contains 100-300 kb.
Contains ORI point
Contains two replication forks
In eukaryotic there are numerous replicons
19.11.2014 28
Peculiarities of replication in eukaryotes
In nucleus replication is carried down by DNA-polymerases a, d
In mitochondria replication is done by DNA-polymerase g.
Chromosomal DNA contains many points ORI;
Replication speed – 20-100 bases/second;
Human nuclear genome contains 60000 ORI points at every 100-150 kb.
Replication of nuclear human DNA is carried out in 6-9 hours19.11.2014 29
Example: If 1st chromosome will contain 1 ori point,
replication will take place during 15 hours:
Contains 260 000 000 bp;
Each replication fork will contain 130 000 000
bp
Replication speed - 100 bp/sec;
19.11.2014 30
11/19/2014
11
Peculiarities of replication of telomeres
Telomeres:
• Sequences of nucleotides at the end of chromosomes;
• (GGGTTAA)n;
• Form loops;
Telomerase:
• DNA-polymerase RNA-dependent;
• Contains snRNA – template for synthesis of telomeric DNA.
19.11.2014 31
Replication of
telomeres
19.11.2014 32
Werner Patient
19.11.2014 33
11/19/2014
12
26 years old
30 years old
Doctor and patient –30 years old 45 years old
19.11.2014 34