Summary
Summary Molecular Genetics
- Course
- Institution
This is a summary of all the lectures of molecular genetics. It is edited with highlights and additional pictures that will help to understand everything better!
[Show more]
1 review
By: roosklaasen • 1 year ago
Seller
Follow
maud91
Reviews received
Content preview
Molecular geneticsLecture 1:
Metabolome → Set of all metabolites in an
organism:
- Genome → All genetic information of
an organism
- Trascuotome → Set of all RNA
transcripts from a genome
- Proteome → Set of all proteins
expressed by an organism
How does the genome replicate?
DNA replication is semiconservative
● Watson & Crick (1953)
○ Semiconservative replication
○ Each “parent” DNA strand produces a new “daughter” strand
● Topological problem
○ How to unwind the DNA
○ Semiconservative, conservative and dispersive replication mechanisms
● Matthey MEselson & Franklin Stahl (1958)
○ Meselson-Stahl experiment
The topological problem
The topology of DNA is defined by how the two DNA strands are intertwined. It plays an
important role in processes such as replication, recombination, transcription, ect.
Topoisomerase → enzymes that catalyze changes in the topological state of DNA by cutting
DNA strands. They relax positive and negative supercoils
- Type I Topoisomerase (singles-strand break)
- Type II topoisomerase (double-strand break)
- Gyrase type II → enzyme that introduces negative supercoils. It relaxes and
prevents overwinding during DNA replication
Initiation of DNA replication
Replication starts at the origin of replication (ori), where DNA strands are separated generating
a replication bubble that contains two replication forks, where replication occurs
, - Bacterial circular chromosomes contain a single ori
- Eukaryotic linear chromosomes have multiple ori. (e.g. human chromosome contain
30.000 and 50.000
A replicon is a unit of the genome that is replicated by a single origin of replication (e.g. one
replicon is bacteria, multiple in eukaryotes)
OriC of E. coli: Methylation regulates initiation
- OriC is 245 bp long and contain multiple recognition sites for
DNA binding proteins and enzymes
- Methylation of the OriC regulates initiation of replication
- OriC contains 11 copies of a palindromic sequence
(GATCCTAG). Dam methylase enzyme methylates the
adenines of the sequence
- When both DNA strand are methylated, oriC is active and
replication starts
- Hemimethylated origins (one strand methylated and other not)
inhibit initiation of replication, only fully methylated origins start
DNA replication (regulation system)
- When OriC is fully methylates, 6 proteins are involved in the formation of the replication
forks and the initiation of replication
1) DnaA is the initiator protein, and it is activated when it is bound to ATP.
DnaA-ATP binds in the fully methylated oriC. First in the high affinity sites then
the DNA wraps around DnaA and it binds to the low affinity sites that are AT rich,
It twists and melts the helix with the help of HU
2) Two DnaB/DnaC complexes, DnaB is an ATP hydrolysis dependent 5’-3’
helicase. It unwinds the DNA by breaking the hydrogen bonds between the
nucleotides. DnaC is a chaperone. They form the two replication forks
3) Gyrase (type II topoisomerase) relaxes DNA supercoils
4) SSB (single strand binding protein) stabilizes DNA, keeps the replication bubble
open, protects against degradation of ssDNA from DD-specific-nucleases
DNA polymerases
DNA-dependent DNA polymerase → multiple DNA polymerase activities in prokaryotes and
eukaryotes involved in different processes (DNA repair, replication, etc. But they all have these
common features:
1) DNA synthesis (5’ - 3’ direction)
2) DNA polymerases cannot initiate synthesis of DNA de novo, they need a primer with a
free 3’ - OH end
3) Proofreading error-control system. Exonuclease activity (3’ - 5’ direction)
DNA polymerase I contain both 3’ - 5’ and 5’- 3’ exonuclease activity. There are at least 14
different DNA polymerases in eukaryotes, but polymerase alpha, beta, gamma and delta have
major roles during replication and have been studies well
,Priming and semi-discontinuous replication
● DNA replication cannot synthesize DNA de novo: Primase is a DNA dependent RNA
polymerase that synthesizes RNA primers of ~ 10 nt long
- Eukaryotes: only one enzyme is required (DNA polymerase alpha has both
activities)
- Prokaryotes: Two different enzymes are required (primase and DNA polymerase
III)
● Leading strand is synthesized
continuously and the lagging strand
discontinuous. Therefore the DNA
replication is discontinuous
● Leading strand primes only once,
lagging strand very often synthesizes Okazaki fragments (1000 - 2000 nt in bacteria, ~
200 nt in eukaryotes) formed by RNA primer + elongated DNA (hybrid oligo)
● Finally RNA primers are degraded, gaps are filled with DNA by DNA polymerase and all
the fragments are ligated together by a ligase enzyme
Joining of adjacent Okazaki fragments in E. coli
- DNA polymerase III has only 3’ -5’ exonuclease
activity. It stops synthesizing DNA when it finds a
primer
- DNA polymerase I has both 3’ - 5’ and 5’ - 3’
exonuclease activities. It degrades the primer with the
5’ - 3’ exonuclease activity and synthesizes new
complementary DNA simultaneously
- DNA ligase ligates the adjacent fragments generating
the lagging strand
Joining of Okazaki adjacent fragments in eukaryotes
Two-step process
- DNA polymerase delta and helicase displace the
primer, creating a 5’ flap. Simultaneously the DNA polymerase fills the gap
- Flap endonuclease I (FEN1) cleaves the flap, removing the primer
- DNA ligase ligates the adjacent fragments
Elongation of DNA replication
Progress at the replication fork: the replisome
- In E. coli the progression of the replication fork is maintained by these enzymes:
Helicase, Gyrase, SSB, Primase, DNA polymerase I and III, Ligase
- Eukaryotes are complex organisms, many other proteins are involved during elongation
but the process is similar
, How does the lagging strand keep the same synthesis rate in both strands?
→ The replisome (a complex of two DNA polymerases + associated proteins) keep both DNA
polymerase linked and assures a parallel synthesis of the leading and lagging strands
→ Loop structures of the lagging strand allows DNA synthesis in the same direction of the
replication fork
The replisome: structure
● The replisome is a multi proteins structure that
assembles at the replication fork to undertake synthesis
of DNA. It contains:
○ 2x DNA polymerase (for lagging and leading
strand)
○ 2x dimerizing subunit T that link DNA
polymerases together
○ 2x sliding clamps form beta-rings that encircles the DNA and ensure contact
between DNA and DNA polymerase
○ Clamp loader (group of 5 proteins) places the clamp on DNA, keeping all the
structure together
● DNA polymerases have different abilities. Each polymerase synthesizes one DNA
strand. DNA polymerase in leading strand remains associated to the DNA template,
DNA polymerase in lagging strand must repetitively associate / dissociate from DNA
The replisome: movement
https://www.youtube.com/watch?v=IjVLhoyfGAM
Termination of DNA replication
Termination of replication in E. coli
- DNA replication is bidirectional and start at OriC
- Replication forks meet and halt in a region halfway from the origin → the replication fork
trap
- At both sites of termination region there are five Ter sites (23 bp) that are recognized by
Tus (terminator utilization substance) proteins
- Tus proteins bind to DNA in a specific orientation and stops the replication fork
→ Eukaryotic termination is more complex (larger linear chromosomes, multiple replicons)
The end problem
Linear chromosomes could become shorter after replication because one of these reasons
a) Final Okazaki fragment cannot be primed (primase doesn’t have space to add a primer)
b) The primer of the last Okazaki fragment is at the very last 3’ extreme. It cannot be
removed and synthesized by DNA polymerase
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller maud91. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $7.43. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
81113 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now