DNA replication. - proteins for initiation of replication; - proteins for polymerization of nucleotides.

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1 DNA replication Replication represents the duplication of the genetic information encoded in DNA that is the crucial step in the reproduction of living organisms and the growth of multicellular organisms. Replication is semiconservative so that each new DNA double helix consists of one parental template strand hydrogen bonded along its entire length by basepairing to a newly synthesized strand (Fig. 1). Fig. 1. Model of replication - proteins for initiation of replication; - proteins for polymerization of nucleotides. Components required for replication: - DNA as template; - deoxyribonucleoside triphosphates (dntp) for synthesis of DNA; - ribonucleoside triphosphates (NTP) for synthesis of primers; - proteins for unwinding the doublehelix of DNA; In the process of replication participate a lot of proteins, including enzymes: - DNA helicases - enzymes that unwind DNA to facilitate separation of the two strands of the duplex. - Primase a RNA-polymerase that synthesizes the short RNA primers for DNA replication using DNA as a template. - Topoisomerases enzymes that catalyse the interconversion of different topological isomers of DNA that involves the transient breakage of one (type I) or both strands (type II) of DNA and can result in the removal of negative or positive supercoils from DNA or the introduction of negative supercoils. - DNA-polymerazes enzymes that synthesis new strands of DNA in the direction 5-3 on the bases of templates. All polymerases can only continue strands, and for initiation of activity need free 3 ends. Some DNA-polymerases have also exonuclease activity: can remove nucleotides from the end in the direction 5-3 or DNA-ligases enzymes that bind the fragments of DNA with 3-5 phosphodiester bonds. - SSB protein bind single-stranded DNA regions. Replication initiates at specific replication origins (ORI), to generate two replication forks, which are elongated bidirectionally by multienzyme replication complexes - the replisome. Prokaryotic circular DNAs are replicated from a single origin, but eukaryotic DNAs have multiple origins of replication so that the large chromosomes can be replicated sufficiently rapidly. A region or unit of a chromosome served by a single origin of replication is called replicon. Mechanisms of replication The two strands of a DNA double helix are antiparallel, that is, they run in opposite directions so that the terminal 5'PO 4 of one strand is opposite the terminal 3'OH of the other. However, DNA polymerases can only add nucleotides to the 3'OH group of a polynucleotide chain. These constraints are overcome by the use of RNA primers and a semi-discontinuous

2 mode of synthesis. With rare exceptions, DNA chains are initiated by synthesis of short RNA primers, which are initiated de novo by RNA polymerases known as primases using the DNA strand as a template. These provide a 3' hydroxyl group from which DNA polymerases can synthesize new DNA. Since the strands are antiparallel and DNA polymerases can only add to the 3' end, synthesis of both strands at a single replication fork requires two mechanisms. One strand, the leading strand, is synthesized continuously by the repeated addition of nucleotides to its 3' end, whereas the other lagging strand is synthesized discontinuously in segments called Okazaki fragments which are about 1000 nucleotides long in bacteria or 150 nucleotides in eukaryotes. Gaps between the fragments are subsequently filled to form the second continuous DNA strand (Fig. 2). Mechanism of replication DNA polymerases DNA polymerases polymerize deoxyribonucleoside triphosphates into DNA by a condensation reaction that forms a phosphodiester bond linking the 3-OH of the sugar component of one nucleotide and the 5-OH of the sugar of the next with the release of pyrophosphate. There are several types of DNA polymerase in any organism. In Escherichia coli, DNA polymerase III synthesizes both leading and lagging strands. The gaps between Okazaki fragments are filled by DNA polymerase I. In eukaryotes, chromosomal DNA is replicated by three DNA polymerases α, δ, and ε. Polymerase α contains an integral primase. Polymerase α is required for lagging strand synthesis and possibly for the initial priming of leading strands too. Polymerase δ is required for leading strand synthesis. Mitochondrial DNA is synthesized by polymerase γ. Most DNA polymerases, but probably not polymerase α, contain a proofreading exonuclease which excises misincorporated bases, increasing the fidelity of template copying. Initiation of replication Prokaryotes initiate DNA replication at unique sites, called origins of replication. In eukaryotes multiple origins are used, so that eukaryotic chromosomes are replicated by many replication forks simultaneously. 2

In prokaryotes and some animal viruses, and presumably elsewhere, replication origins are recognized by sequence-specific binding proteins, which can locally unwind a specific region of the double

3 In prokaryotes and some animal viruses, and presumably elsewhere, replication origins are recognized by sequence-specific binding proteins, which can locally unwind a specific region of the double helix allowing replication to initiate. Initiation occurs in some steps: - recognition of ORI by special proteins; - DNA-helicase unwinds DNA; - Primase synthesis a short fragment of RNA primer; - DNA-polymerase adds new nucleotides at 3 end (Fig. 3). All of proteins which participate at initiation form the promosome. Unidirectional replication occurs in some phages and some prokaryotic circular DNAs replicate by a rolling circle mechanism (Fig. 4). Fidelity of DNA replication is ensured by proofreading and DNA repair mechanisms. Regulation of DNA replication is a critical control point in cell proliferation. Fig. 3. Initiation of replication Fig. 4. Rolling circle mechanism of replication 3

4 Termination and telomeres When two converging replication forks meet, their nascent strands are joined. DNA topoisomerase II is required to unwind the two progeny DNA molecules from around each other in these final stages. The ends of the long linear chromosomes of eukaryotes, called telomeres, are replicated by a different mechanism. They consist of many copies of a short repeating sequence which are added by the enzyme telomerase. This enzyme contains an RNA template which it copies into DNA to complete the chromosome ends (Fig. 5). Mitochondrial DNA has two ORI one for light strand (L) and the other for heavy starnds (H). Fig. 5. Replication of telomeres DNA repair DNA repair represents a range of cellular responses associated with restoration of primary structure of DNA. Mechanisms of alteration of DNA molecules: - base substitution during replication - base changes resulting from chemical instability of bases - alterations resulting from the action of other chemical and environmental agents. The possible defects in DNA molecules: - An incorrect base in one strand cannot form hydrogen bond with the corresponding base in the other. Defect can result from replication errors or Deamination of C to U, followed by replacing of U by T in subsequent rounds of replication. Fig. 6. Formation of pyrimidinic dimers - Missing bases depurination - Altered bases Alkylating agents (used in cancer treatment) react with G and weak N- glycosidic bond. Chemical and physical agents can break purine and pyrimidine rings. Formation of thymine dimmers (Fig. 6). - Single-strand breaks. Peroxides, Fe 2+, Ca 2+, Ionizing radiation can attack the phosphodiester bonds 4

DNases present in cells make phosphodiester scissions. - Double-strand breaks. Highly ionizing radiations can produce numerous single strand breaks and as result double-strand breaks.

Enzymes synthesis DNA without fidelity of replication. Types of reparation Photoreactivation - direct repair, when enzymatic cleavage of thymine dimers is activated by visible light.

5 DNases present in cells make phosphodiester scissions. - Double-strand breaks. Highly ionizing radiations can produce numerous single strand breaks and as result double-strand breaks. Mechanisms of DNA repair - Excision repair excision of damaged bases - Recombination repair reconstruction of DNA from undamaged fragments - SOS repair in prokaryotic cells. Enzymes synthesis DNA without fidelity of replication. Types of reparation Photoreactivation - direct repair, when enzymatic cleavage of thymine dimers is activated by visible light. Photolyase was discovered in different species, but is active only in bacteria. A Fig. 7. A - Base excision repair (BER); B - Nucleotide excision repair (NER) B Repair of alkylation damages by MGMT O 6 -methyl-guanine DNA methyltransferase (in human). 20% of human tumor cell have reduced MGMT activity. Base excision repair (BER) reparation of single nucleotide damage (Fig. 7.A). DNA-glycosylase removes the damaged (modified, fragmented) base from DNA AP-lyase makes an excision at 3 end (AP apurinic/apyrimidinic sites) AP-hydrolase incises at 5 end DNA-polymarease fills in the gap beginning with 3 end DNA-ligase seals the nick. Nucleotide excision repair (NER) reparation of bulky DNA damages (Fig. 7.B) 5

A complex of proteins (XPA-PRA) recognizes the damaged fragment Excinuclease cuts the 5 th phosphodiester bond 3 and 24 th phosphodiester bond 5 to the lesion (single-strand incisions) The gap is

6 A complex of proteins (XPA-PRA) recognizes the damaged fragment Excinuclease cuts the 5 th phosphodiester bond 3 and 24 th phosphodiester bond 5 to the lesion (single-strand incisions) The gap is filled by DNA-polymerases δ and ε DNA-ligase seals the nick. Mismatch repair (MMR) reparation of misincorporations during replication and the mismatches resulted of deamination of 5-methylcytosine to uracil An unknown enzyme excises the mismatch bases The alkylating agents induced expression of DNA-polymerase β which fills in the gap DNA-ligase seals the nick. Recombination repair (Fig. 8) - Excision of damaged fragment of DNA - Transport of homologous fragment from another molecule - Legation of the fragments. Fig. 8. Recombination repair 6

Welcome to Class 18! Lecture 18: Outline and Objectives. Replication is semiconservative! Replication: DNA DNA! Introductory Biochemistry!

Welcome to Class 18! Lecture 18: Outline and Objectives. Replication is semiconservative! Replication: DNA DNA! Introductory Biochemistry! Lecture 18: Outline and Objectives Welcome to Class 18! Introductory Biochemistry! l DNA Replication! l DNA polymerase! l the enzymatic reaction! l proofreading and accuracy! l DNA synthesis! l origins