Molekulární biologie(KBC/MBIOG)

Ivo FrébortAlberts et al. (2008) Molecular Biology of the Cell, 5th ed.

Garland Science, New York

• 3. Replication, Repair, and Recombination of DNA

Different rates of protein evolution – natural selection

Semiconservative replication of DNA

The chemistry of DNA synthesis

DNA replication fork

DNA polymerase

E. coli Pol I (928 aa, 109 kDa) monomer

Editing by DNA Pol I

Exonucleic proofreading by DNA polymerase

RNA primer synthesis(DNA primase)

The reaction catalyzed by DNA ligase

DNA helicase – opening the replication fork

Single-strand binding proteins straighten the unwound helix

Sliding clamp prevents DNA polymerase from dissociation

E. coli Human Pol III PCNA protein

Loading and unloading of DNA polymerase on the lagging strand

Procaryotic replication fork

Moving replication fork

Moving replication fork, part II

Moving replication fork, part III

Strand directed mismatch repair in eucaryotes

MutS

The „winding problem“ of the DNA replication

DNA topoisomerase Ihelps rotation by reversible nicking of one strand

DNA topoisomerase II makes transient double-strand break

Single origin of DNA replication in procaryotes

Methylation of the E. coli origin of replicationLag phase (10 min) – mismatch repairing system

Initiation and completion of DNA replicationReplication bubble

Mammalian DNA replication fork

Eucaryotic chromosomes contain multiple origins of replication

DNA replication in eucaryotes takes place only during the S-phase of cell cycle

Mammals 8h, yeast 40 min

DNA replication in eucaryotes and the cell cycle

Identification of replication origins (ARS) in yeast

Large „Origin Recognition Complex“ initiates the replication

Yeast

Human

Nucleosome assembly during replication

Inheritance of histone modification

Telomere replication

Control of the lenght of telomeres

DNA repair

Spontaneous alterations requiring DNA repair

Red – oxidative damageBlue – hydrolysisGreen - methylation

Depurination and deamination

Pyrimidine base dimerization by UV light

Formation of mutations during replication of damaged DNA

Two major DNA repair pathways

Recognition of unusual nucleotide by base-flipping(DNA glycosylases)

Deamination of DNA nucleotides. Why not U in DNA?

Specific DNA glycosylase

T-G pairing glycosylase

Repair of double strand breaks

Non-homologous end-joining by Ku protein

„Quick and dirty“ repair

General (homologous) recombination

DNA hybridization, the principle of recombination

Repair of single strand breaks by homologous recombinantion

DNA synapsis catalyzed by RecA protein in E. coli

Rad 51 and Rad52 in humans

Flawless repair of double strand breaks by homologous recombinantion

Holliday Junction

Crossovers by homologous recombination in meiosis

Homologous recombination in meiosis

Gene conversion by mismatch correction and proofreading

Differences in general recombination in mitotic and meiotic cells

Site-specific recombination

Some mobile genetic elements of bacteria –DNA-only transposons

Cut-and-paste transposition

The life cycle of a retrovirus

Reverse transcriptase

Transposition by a retrovirus (such as HIV) or a retroviral-like retrotransposon

Transposition by a nonretroviral retrotransposon

Conservative site-specific recombination

Insertion of lambda DNA into bacterial chromosome

The life cycle of bacteriophage lambda

Conservative site-specific recombination can be used to turn genes on and off (mouse)