[CHEM200] Biosynthesis of Nucleic Acids (Replication) Reviewer

ULBIS, CHRISTINE MARIE HONG (SEAT # 36) 3BIOLOGY-6 FLOW OF GENETIC INFORMATION IN THE CELL o Replication – process of duplication of DNA o Transcription – process of formation of RNA on a DNA template o Translation – process of protein synthesis in which the amino acid sequence of the protein reflects the sequence of bases in the gene that codes for that protein o Reverse Transcriptase – the enzyme that directs the synthesis of DNA on an RNA template  Sequence of bases in DNA encodes genetic information.  The duplication of DNA, giving rise to new DNA molecule with the same base sequence as the original, is necessary whenever a cell divides to produce daughter cells.  The actual formation of gene products requires RNA.  The base sequence of DNA is reflected in the base sequence of RNA.  The three kinds of RNA are involved in the biosynthesis of proteins.  Of the three, messenger RNA (mRNA) specifies the identity of one amino acid in a manner directed by the genetic code.  The flow of genetic information is DNA->RNA>protein.  The only major exceptions are some viruses (called retroviruses) in which RNA, rather than DNA, is the genetic material.  In those viruses, RNA can direct its own synthesis as well as that of DNA.  Not all viruses in which RNA is the genetic material are retroviruses, but all retroviruses have a reverse transcriptase. REPLICATION OF DNA Nucleases – enzymes that hydrolyze a nucleic acid and are specific or DNA or RNA 

The process which one double-helical DNA duplicates molecule to produce two doublestranded molecules is complex.  The cell faces three important challenges in carrying out the necessary steps. 1. Separating the two DNA strands. The two strands of DNA are wound around each other in such a way that they must be unwound if they are to be separated. In addition to achieving continuous unwinding of the double helix, the cell also must protect the unwound portions of DNA from the action of nucleases that preferentially attack single-stranded DNA. 2. Synthesizing of DNA from the 5’ to the 3’ end. Two antiparallel strands must be synthesized in the same

direction on antiparallel templates. In other words, the template has one 5’>3’ strand and one 3’->5’ strand, as does the newly synthesized DNA. 3. Guarding against errors in replication. Ensuring that the correct base is added to the growing polynucleotide chain. Semiconservative Replication - the mode in which DNA reproduces itself, such that one strand comes from parent DNA and the other strand is newly formed  DNA replication involves separation of the two original strands and production of two new strands with the original strands as templates.  Each new DNA molecule contains one strand from the original DNA and one newly synthesized strand. Density-Gradient Centrifugation – the technique of separating substances in an ultracentrifuge by applying the sample in the top of a tube that contains a solution of varying densities Origin of Replication – the point at which the DNA double helix begins to unwind at the start of replication  New polynucleotide chains are synthesized using each of the exposed strands as a template.  Two possibilities exist for growth of the new strands: synthesis can take place in both directions from the origin of replication, or in one direction only. Replication Forks – in DNA replication, the points at which new DNA strands are formed  A “bubble” of newly synthesized DNA between regions of the original DNA is a manifestation of the advance of the two replication forks in opposite directions.  The bubbles grow larger and eventually merge, giving rise to two complete daughter DNAs. This biological growth of both polynucleotide chains represents net chain growth.  Both new polynucleotide chains are synthesized in the 5’->3’ direction. DNA POLYMERASE Semidiscontinuous DNA Replication  All synthesis of nucleotide chains occurs in the 5’->3’ direction from the perspective of the chain being synthesized. This is due to the nature of the reaction of DNA synthesis.  The last nucleotide added to a growing chain has a 3’-hydroxyl on the sugar.  The incoming nucleotide has 5’triphosphate on its sugar.  The 3’-hydroxyl group at the end of the growing chain is a nucleophile. It attacks the phosphorus adjacent to the sugar in the nucleotide to be added to the growing chain, leading to the elimination

of the pyrophosphate and the formation of a new phosphodiester bond. DNA Strands in Opposite Direction  One newly formed strand (leading strand) is formed continuously from its 5’ end to its 3’ end at the replication fork on the exposed 3’ and 5’ template strand.  The other strand (lagging strand) is formed semi discontinuously in small fragments, sometimes called Okazaki fragments.  The 5’ end of each of these fragments is closer to the replication fork than the 3’ end.  The fragments of the lagging strand are then linked together by an enzyme called DNA ligase. DNA Polymerase from E.coli  The first DNA polymerase discovered was found in E.coli. At least five DNA polymerases are present.  Polymerase I (Pol I) consists of a single polypeptide chain.  Polymerase II (Pol II) is not required for replication; rather, it is strictly a repair enzyme.  Polymerase III (Pol III) shares common subunits in Pol II.  It consists of a core enzyme responsible for polymerization and 3’ exonuclease activity-consisting of a-, e-, and 0-subunits-and a number of other subunits, including a dimer of a-subunits responsible for DNA binding, and the y-complex-consisting of y-, 8-,8’,x-, and y-subunits which allows the b-subunits to form a clamp that surrounds the DNA and slides along it as a polymerization proceeds.  It has the highest turnover number and a huge processivity compared to polymerase I and II.  Pol IV and Pol V are repair enzymes and both are involved in a unique repair mechanism called the SOS response.  Considerations regarding polymerases: 1. Speed of the synthetic reaction (turnover number) 2. Processivity – the number of nucleotides incorporated in a growing DNA chain before the DNA polymerase disassociates from the template DNA  DNA polymerase – catalyzes the successive addition of each new nucleotide to the growing chain  This enzyme forms DNA from deoxyribonucleotides on a DNA template  Primer – in DNA replication, a short stretch of RNA hydrogen-bonded to the template DNA to

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which the growing DNA strand is bonded at the start of synthesis DNA polymerase reaction requires all four deoxyribonucleoside triphosphates-dTTP, dGTP, and dCTP. Proffreading – the process of removing incorrect nucleotides when DNA replication is in progress  It is done one nucleotide at a time. Repair – the enzymatic removal of incorrect nucleotides from DNA and their replacement by correct ones

PROTEINS REQUIRED FOR DNA REPLICATION Replisome – a complex of DNA polymerase  It is the RNA primer, primase and helicase at the replication fork. Two questions arise in separating the two strands of the original DNA so that it can be replicated. 1. How to achieve continuous unwinding of the double helix? 2. How to protect single-stranded stretches of DNA that are exposed to intracellular nucleases as a result of the unwinding? Supercoiling and Replication o DNA gyrase – (class II topoisomerase)  It is an enzyme that introduces supercoiling into closed circular DNA.  It catalyzes the conversion of relaxed, circular DNA with a nick in one strand to the supercoiled form with the nick sealed that is found in normal prokaryotic DNA.  A light unwinding of the helix before the nick is sealed introduces the supercoiling.  The energy require for the process is supplied by the hydrolysis of ATP. 

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In replication, the role of the gyrase is different.  The prokaryotic DNA is negatively supercoiled in its natural state; however, opening the helix during replication would introduce positive supercoils ahead of the replication fork.  DNA gyrase fights positive supercoils by putting negative supercoils ahead of the replication fork. Helicase – a protein that unwinds the double helix of DNA in the process of replication  It promotes unwinding by binding at the replication fork  This includes DnaB protein and rep protein Single-Strand Binding Protein (SSB) – in DNA replication, a protein that protects exposed single-strtand sections of DNA from nucleases  It stabilizes the single-stranded regions by binding tightly to these portions of the molecule

 This protects single-stranded regions which are very susceptible to degradation from hydrolysis by the nucleases Primase Reaction  RNA serves as a primer in DNA replication.  A primer in DNA replication must have a free 3’-hydroxyl to which the growing chain can attach, and both RNA and DNA can provide this group.  Primase – the enzyme that makes a short section of RNA to act as a primer for DNA synthesis  It responsible for copying a short stretch of the DNA template to produce the RNA primer sequence.  Primosome – the complex at the replication fork in DNA synthesis  It consists of the RNA primer, primase, and helicase. Synthesis and Linking of New DNA Strands  The synthesis of two new strands of DNA is begun by DNA polymerase III.  The newly formed DNA is linked to the 3’hydroxyl of the RNA primer, and synthesis proceeds from the 5’ end to the 3’ end on both the leading and the lagging strands.  As the replication fork moves, the RNA primer is removed by polymerase I, using its exonuclease activity.  The primer is replaced by deoxynucleotides, also by DNA polymerase I, using its polymerase activity.  None of the DNA polymerase can seal the nicks that remain; DNA ligase is the enzyme responsible for the final linking of the new strand. Summary of DNA Replication in Prokaryotes 1. DNA synthesis is bidirectional. Two replication forks advance in opposite directions from an origin of replication. 2. Two direction of DNA synthesis is from the 5’ end to the 3’ end of the newly formed strand. One strand (the leading strand) is formed continuously, while the other strand (the lagging strand) is formed discontinuously. On the lagging strand, small fragments of DNA (Okazaki fragments) are subsequently linked. 3. Five DNA polymerase have been found in E. coli. Polymerase III is primarily responsible for the synthesis of new strands. The first polymerase enzyme discovered, polymerase I, is involved in synthesis, proofreading, and repair. Polymerase II, IV and V function as repair enzymes under unique conditions. 4. DNA gyrase introduces a swivel point in advance of the movement of the replication fork. A helix-destabilizing protein, a helicase, binds at the replication fork and promotes unwinding. The exposed single-stranded

regions of the template are stabilized by a DNA-binding protein. 5. Primase catalyzes the synthesis of an RNA primer. 6. The synthesis of new strands is catalyzed by Pol III. The primer is removed by Pol I, which also replaces the primer with deoxynucleotides. DNA ligase seals the remaining nicks. 

In E. coli, the polymerizing part of Pol III is a dimer of the B-subunit, and it forms a closed ring, called a sliding clamp, around the DNA chain.  This is a complication for the entire process of replication as somehow this ring has to get around the DNA.  The clamp cannot spontaneously surround the DNA chain because the dimer is a closed circle. Instead, the part of the Pol III enzyme that is called the clamp loader opens the sliding clamp and inserts the DNA chain.  All clamp loaders are pentameric enzymes who are members of a family of ATPases called the AAA+ superfamily.

PROOFREADING AND REPAIR  DNA replication takes place only one each generation in each cell. It is essential that fidelity of the replication process be as high as possible to prevent mutations, which are errors in replication. o Mutations – changes in DNA, causing subsequent changes in an organism that can be transmitted genetically  Errors in replication occur spontaneously only once in every 109 to 1010 base pairs. o Proofreading – refers to the removal of incorrect nucelotides immediately after they are added to the growing DNA during the replication process  DNA polymerase has three active sites.  Pol I can be cleaved into two major fragments.  One of them (the Klenow fragment) contains the polymerase activity and the proofreading activity.  The other contains the 5’->3’ repair activity. 

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Errors in hydrogen bonding lead to the incorporation of an incorrect nucleotide into a growing DNA chain once in every 10 4 to 105 base pairs. DNA polymerase I uses its 3’ exonuclease activity to remove the incorrect nucleotide. Replication resumes when the correct nucleotide is added, also by DNA polymerase I. During replication, a cut-and-patch process catalyzed by polymerase I takes place.  The cutting is the removal of the RNA primer by the 5’ exonuclease function of the polymerase, and the patching is the incorporation of the required

deoxynucleotides by the function of the same enzyme.

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Nick Translation – a type of DNA repair that involves polymerase I using its 5’ to 3’ exonuclease activity to remove primers or replace damaged nucleotides Mutagens – agents that bring mutation; such agents include radiation & chemical substances that alter DNA  Ultraviolet light creates pyramidine dimers. The pi elections from two carbons on each of two pyramidines form a cyclobutyl ring, which distorts the normal shape of the DNA and interferes with replication and transcription.  Chemical damage, which is often cause by free radicals can lead to a break in the phosphodiester backbone of the DNA strand.

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When damage has managed to escape the normal exonuclease activities of DNA polymerase I and III, prokaryotes have a variety of other repair mechanisms at their disposal.

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Mismatch Repair – type of DNA repair that begins when repair enzymes find two bases that are incorrectly paired  Area with the mismatched is removed and DNA polymerases replicate the area again.  If there is a mismatch, the challenge for the repair system is to know which of the two strands is the correct one.

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Base-Excision Repair – a type of DNA repair that begins with an enzyme removing a damaged base, followed by removal of the rest of the nucleotide 1) A base that has been damaged by oxidation or chemical modification is removed by DNA glycosylase, leaving an AP site, so called because it is apurinis or apyrimidinic (without purine or pyramidine). 2) An AP endonuclease then removes the sugar and phosphate from the nucleotide. 3) An excision exonuclease then removes several more bases. 4) Finally, DNA polymerase I fills in the gap, and DNA ligase seals the phosphodiester backbone.

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Nucleotide-Excision Repair – type of DNA repair in which damaged or deformed DNA is repaired by removal of a section of DNA containing the damage 1) Large section of DNA containing the lesion is removed by ABC excinuclease. 2) DNA polymerase I and DNA ligase then work to fill the gap.

DNA RECOMBINATION Genetic Recombination – general term for several processes whereby genetic information is rearranged

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At a molecular level, genetic recombination is the exchange of one DNA sequence with another or the incorporation of a DNA sequence into another.  Homologous Recombination – the genetic recombination between homologous DNA sequences  It also termed general recombination because the enzymes that mediate the exchange can use essentially any pair of homologous DNA sequences.  Nonhomologous Recombination – the genetic recombination between very different nucleotide sequences Recombination does not occur randomly in a chromosome. o Hot Spots – areas on a chromosome that are likely to have recombination events Recombination occurs by the breakage and reunion of DNA strands so that physical exchange of DNA parts takes place o Holliday Model – this model was deduced 1964 by Robin Holliday  It is the model for how recombination occurs between the homologous chromosomes. 1) Two homologous DNA segments align. In eukaryotes, this is called chromosome pairing. 2) A nick occurs at the same place on two homologous strands. 3) The DNAs on the two strands then swap places, or cross over, at the nick by the process of strand invasion. 4) The crossing over can then proceed down each strand of DNA. 5) The branch migration leads to strand exchange between the two homologous DNA pieces. This leads to exchange of genes and traits caused by them. Recombination is a critical process during meiosis.  Segregation of chromosomes during formation of gametes is quite inaccurate, with estimates indicating that abnormal chromosomes numbers in gametes, called aneuploidy, occur in 10%-25% of all conceptions.

EUKARYOTIC DNA REPLICATION  Eukaryotic chromosomes accomplish DNA synthesis by having replication begin at multiple origins of replication, also called replicators. o Replicators are specific DNA sequences that are usually between gene sequences.

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The zones where replication is proceeding are called replicons, and the size of these varies with the species.

Only chromosomes from cells that have reached the G1 phase are competent to initiate DNA replication.  Many proteins are involved in the control of replication and its link to the cell cycle. The first proteins involved are seen during a window of opportunity that occurs between the early and late G1 phase.  Replication is initiated by a multi-subunit protein called the origin recognition complex (ORC).  It is a protein complex bound to DNA throughout the cell cycle that serves as an attachment site for several proteins that help control replication.  The next protein to bind is an activation factor called the replication activator protein (RAP).  It is the protein whose binding prepares for the start of DNA replication in eukaryotes.  After the activator protein is bound, replication licensing factors (RLFs) can bind.  They are proteins required for DNA replication in eukaryotes.  Replication cannot proceed until they are bound.  Some RLF proteins have been found to be cystolic.  They have access to he chromosome only when the nuclear membrane dissolves during mitosis.  After RLFs bind, the DNA competent for replication.

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The activation of cylin-CDKs serves both to initiate DNA replication and to prevent formation of another pre-RC. In the G2 phase, the DNA has been replicated. During mitosis, the DNA is separated into the daughter cells. At the same time, the dissolved nuclear membrane allows entrance of the licensing factors that are produced in the cytosol so that each daughter cell can initiate a new round of replication.

Eukaryotic DNA Polymerases  At least 19 different polymerases are present in eukaryotes.  The five-best studied polymerases are called a, B, y, 8, and E.  a, B, 8 and E are found in the nucleus.  Y form occurs in mitochondria. 

Polymerase a has the most subunits. It has the ability to make primers, but lacks a 3’->5’ proofreading activity that has low processivity.  After making the RNA primer, Pol a, adds about 20 nucleotides and is then replaced by Pol 8 and E.

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Polymerase 8 is the principal DNA polymerase in eukaryotes. It interacts with a special protein PCNA (proliferating cell nuclear antigen).  PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (B). It is a trimer of three identical proteins that surround the DNA.

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The role of DNA polymerase E is less clear, but is suggested to be involved in leading strand replication. It may replase polymerase 8 in lagging strand synthesis DNA polymerase B appears to be a repair enzyme.

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DNA polymerase y carries out DNA replication in mitochondria.

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The combination of the DNA, ORC, RAP, and RLFs constitutes the pre-replication complex (pre-RC).  The complex of DNA, recognition protein (ORC), activator protein (RAP), and licensing factors (RLFs) that makes DNA competent for replication in eukaryotes. When cyclins combine with CDKs, they can activate DNA replication and also block reassembly of a pre-RC after intiation. o Cyclins are proteins that play an important role in control of the cell cycle by regulating the activity of kinases. They are proteins that are produced in one part of a cell cycle and degraded in another. o Cyclin-dependent protein kinases (CDKs) are protein kinases that interact with cyclins and control replication.

THE EUKARYOTIC REPLICATION FORK  DNA replication in eukaryotes is semiconservative.  There is a leading strand with continuous synthesis in the 5’->3’ direction and a lagging strand with discontinuous synthesis in the 5’->3’ direction.  An RNA primer is formed by a specific enzyme in eukaryotic DNA replication and the primase activity is associated with Pol a.  The formation of Okazaki fragments is initiated by Pol a.  After the RNA primer is made and a few nucleotides are added by Pol a, the polymerase disassociates and is replaced by

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Pol 8 and its attached PCNA protein. Another protein, called RFC (replication factor C), is involved in attaching PCNA to Pol 8. The RNA primer is eventually degraded, but since polymerases do not have 5’->3’ exonuclease activity, separate enzymes FEN1 and RNase H1 degrade the RNA.

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Finally, DNA ligase seals the nicks that separate the fragments.

Difference in DNA Replication in Prokaryotes and Eukaryotes Prokaryotes Eukaryotes Five polymerases (I, II, III, IV, V) Five polymerases (a, B, y, 8, E) Functions of polymerases: I is involved in synthesis, proofreading, repair and removal of RNA primers II is also a repair enzyme III is the main polymerizing enzyme IV, V are repair enzymes under usual conditions Polymerases are also exonucleases One origin of replication Okazaki fragments 1000-2000 residues long No proteins complexed to DNA

Functions of polymerases a is a polymerizing enzyme B is a repair enzyme Y is involved in mitochondrial DNA synthesis 8 us the main polymerizing enzyme E is the leading strand replication enzyme Not all polymerases are exonucleases Several origins of replication Okazaki fragments 150-200 residues long Histones complexed to DNA