Dna Structure _ Replication

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DNA Structure & Replication Reference Books

•Lippincotts illustrated review of Biochemistry by Champe and Harvey. •Medical Biochemistry by Baynes and Dominiczak. Structure of deoxyribonuleic acid : -

Cellular nucleic acid exist of two forms. DNA and RNA .About 90% of the nucleic within cells is RNA, and the remainder is DNA. DNA is the storage of genetic information within the cell.

- All the DNA in one human cell (on all 46 chromosomes) is about two meters long ,fits into a cell nucleus which is 2-3 micrometers.The DNA must still be in such a state as to allow for enzymes to replicate the molecule or initiate the production of a protein. The 23 pairs of human chromosomes are estimated to include about 100,000 genes. Each gene codes for ONE protein;

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NUCLEOSIDES -

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Both DNA and RNA contain nucleotides with similar components. In RNA, the sugar component is ribose, as indicated by the name "ribonucleic acid". In DNA, or deoxyribonucleic acid, the sugar component is deoxyribose. The prefix deoxy means that an oxygen atom is missing from one of the ribose Carbon atoms. When a sugar bonds together with a Nitrogen base, This structure is known as a nucleoside. NUCLEOTIDES • The

nucleotides that are the building blocks of nucleic acids are formed by adding a phosphate group to a nucleoside. Nucleotides containing ribose are known as ribonucleotides, and those containing deoxyribose are known as deoxyribonucleotides. • To summarize the structural differences between DNA and RNA: • DNA (deoxyribonucleic acid) • Sugar is deoxyribose • DNA is a polymer of deoxyribonucleotides • Bases are adenine, guanine, cytosine and thymine • RNA (ribonucleic acid) • Sugar is ribose • RNA is a polymer of ribonucleotides. • Bases are adenine, guanine, cytosine and uracil (instead of thymine) •

THE STRUCTURE OF NUCLEIC ACID CHAINS -

Nucleotides are joined together in DNA and RNA by phosphate ester bonds between the phosphate component of one nucleotide and the sugar component of the next nucleotide. More and more nucleotides can be added on by the same process of forming ester bonds until an immense chain is formed. But no matter how long a polynucleotide chain is, one end of the nucleic acid molecule always has a free -OH group on the sugar at the Carbon known as C3' (called the 3' end) and the other end of the molecule always has a phosphoric acid group at C5' (the 5' end).

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There are two types of groove in the double helix of DNA : o Major groove : is more open and exposes the nucleotide base pairs .Binding of proteins to DNA occurs in this groove. o Minor groove : Is more constricted ,being partially blocked by the ribosyl moieties linking the base pairs.

Types of DNA 1- DNA conformation is called B form and is the normal form that present in cells 2- DNA can adopt different configuration in special circumstances when dehydrated, the double helix is more squat in shape and this form is known A form. 3- Another form is called Z form because the polynucleotides backbone zigzags in this double helix is left handed.

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THE WATSON-CRICK MODEL BASE-PAIRING IN DNA :Experiments have shown that DNA samples taken from different cells of the same species have the same proportions of the four bases. Human DNA contains about 30% each of adenine and thymine, and 20% each of guanine and cytosine. The figure is different for other organisms, but the amounts of A and T are always the same, as are the amounts of C and G!

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In 1953, James Watson and Francis Crick proposed a structure for DNA. According to the Watson-Crick model, a DNA molecule consists of two polynucleotide strands coiled around each other in a helical "twisted ladder" structure .The sugar-phosphate backbone is on the outside of the double helix, and the bases are on the inside, so that a base on one strand points directly toward a base on the second strand. The sugarphosphate backbones as the two sides of the ladder and the bases in the middle as the rungs of the ladder.

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The two strands of the DNA double helix run in opposite directions, one in the 5' to 3' direction, the other in the 3' to 5' direction. The term that describes how the two strands relate to each other is known as antiparallel.

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* So what holds the two strands together at the bases? -

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The strands are held together by hydrogen bonds between the nitrogenous bases. In the double helix, adenine and thymine form two hydrogen bonds to each other Similarly, cytosine and guanine form three hydrogen bonds to each other in the double helix. Every base pair contains one purine and one pyrimidine ALWAYS DNA helix to exist in a physically and chemically stable structure. This type of base pairing is called complementary rather than identical. This complementary base pairing in the two strands explains why A/T and G/C always occur in equal amounts. =>

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The cell cycle -

The G1 phase is a period of cell growth that occur prior to DNA replication.

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The phase during which DNA is synthesized or replicated is termed the S phase.

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A second growth phase termed G2 occurs after DNA replication but prior to cell division.

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The mitosis or M phase is the period of cell division. Following mitosis ,the daughter cells either re-enter the G1 phase or enter a quiescent phase termed G0 where growth and replication ceases.

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The passage of cells through the cell cycle is tightly controlled by variety of protein called cyclin_ dependent kinases.

Replication: •Genetic material must be able to be accurately replicated and passed on from one generation to the next. The double helix of DNA suggested the strands can separate and act as template for the formation of a new , complementary strand.

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Basic requirements for DNA synthesis: 1- Substrates: The four deoxynucleotide triphosphate (dNTPs) ,dATP, dGTP ,dCTP, dTTP are needed as substrate for DNA synthesis .Cleavage of high energy phosphate provides the energy for the addition of the nucleotide 2- Template: DNA replication can not occur without a template .Template( nucleic acid strand whose base sequence is copied in a polymerization reaction). A template is required to direct addition of the appropriate complementary nucleotide to the newly synthesised DNA strand. Each strand of parent DNA serves as a template. 3- Primer ( in nucleic acids , a short RNA or single stranded DNA segment that functions as a growing point in polymerization. DNA synthesis can not start without a primer , which prepares the template strand for the addition of nucleotide. Because new nucleotide are added to the 3- of a primer, new synthesis is said to occur in 5- to 3- . 4. Enzymes : The DNA synthesis that occurs during the process of replication is catalyzed by enzyme called DNA-dependant DNA polymerases. The enzymes are DNA dependant by the fact that they require a DNA template. They are called DNA polymerases.

Multiple DNA polymerases with multiple enzymatic activities : The bacteria E-coli contains three separate DNA polymerases. These enzymes are capable of catalyzing other reactions an important role in replication and DNA repair.

Properties DNA polymerases Feature

DNA polymerases 1

5--------3-(exonuclease activity)

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3- ------5-( exonuclease activity) Synthesis rate( nucleotides/min) Replication Repair

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11 -ve +

600 + +

111 -ve +

30 -ve ?

30000 + -ve

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- DNA polymerase 1 was the first DNA polymerase discovered : - A. Function : pol 1 function in the replication of DNA and in the repair of damaged DNA. - B. Structure : pol 1 is a single polypeptide of 103,000 mw - C. Other enzymatic activites : pol1 has two enzymatic acitivtes besides DNA polymerase activity that are important to its cellular function. 1. Proofreading : Pol 1 does not typically add a nucleotide to the growing DNA chain that can not properly base pair with template strand. If a mismatched nucleotide is added , the enzyme halts polymerization .A 3- to 5- exonuclease removes the mismatched nucleotide and polymerization resumes. This activity is called proofreading. 2. Excision _repair : Pol 1 has a 5- to 3- exonuclease acitivity that called excision – repair activity that can - Hydrolytically remove a segment of DNA from 5- end of a strand of duplex DNA. A. One to ten nucleotide segments of DNA can be removed at one time. B. This activity is essential for the removal of primers in DNA replication. C .This activity is essential for the repair of damaged DNA.

- DNA polymerase 11 may be involved in some DNA repair processes .Is a single polypeptide of 90,000 mw. It has proofreading activity ( 3- to 5- exonuclease activity but lacks excision – repair ( 5to 3- exonuclease activity )

- DNA polymerase 111 is the primary DNA polymerase involved in cellular replication. It is structurally complex, is made of 10 subunits polypeptides (422,000 mw). Catalyses leading and lagging strand synthesis. REPLICATION OF DNA ( How is cellular DNA copied? ) DNA replication begins with a partial unwinding of the double helix at an area known as the replication fork. This unwinding is accomplished by an enzyme known as DNA helicase.. This unwound section appears under electron microscopes as a "bubble“and is thus known as a replication bubble.

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DNA Replication -

DNA replication is semi-conservative, one strand serves as the template for the second strand. DNA replication only occurs at a specific step in the cell cycle.

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Stage Activity

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Duration G1 Growth and increase in cell size 10 hr

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S DNA synthesis 8 hr

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G2 Post-DNA synthesis 5 hr

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M Mitosis 1 hr

Replication (higher detail)

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Replication Proteins (Preview) • • • • • •

Helicase Topoisomerase Single strand binding protein DNA polymerase (multiple) Primase DNA ligase

In the Beginning… -

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Problems due to structure o DNA is double-stranded o Template must be single-stranded o Strands must be separated o Separation is difficult due to structure o DNA often in “supercoiled” state.

Helicase:

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Enzyme that separates double-stranded DNA Recognizes action of initiator protein Binds to “origin of replication” and moves, causing strand separation Energy-dependent

Topoisomerase : > • • •

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Relieves stress caused by melting DNA Cleaves DNA and spins around itself to unwind helix Type I cleaves one strand, type II cleaves both Reseals DNA strands after relaxation achieved

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Single Strand Binding Protein :

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> Binds to DNA with no sequence preference  Binds tighter to single strand than double  Keeps separated strands from rejoining

DNA Polymerase : • • • • •

Several forms exist Multiple catalytic functions Synthesizes a DNA strand Uses existing strand as template Can only add to an existing 3’ end

,.;'**********';.,

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Primase Creates a primer for DNA polymerase Template-dependant An RNA polymerase Active briefly at beginning of strand synthesis

DNA Ligase

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• • •

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Joins DNA ends together Forms bond between 5’ PO4 and 3’ Energy requiring reaction

Finishing DNA Synthesis: • • •

DNA polymerase III holoenzyme extends primers made by primase o Stays constantly on leading strand until end o On lagging strand only until previous primer is encountered DNA polymerase I removes primer with 5’-3’ exonuclease and replaces with DNA Ligase joins pol III DNA with pol I DNA

Since each new strand is complementary to its old template strand, two identical new copies of the DNA double helix are produced during replication. In each new helix, one strand is the old template and the other is newly synthesized, a result described by saying that the replication is semi-conservative.

Conservative replication : would occur if , after replication and cell division , the parental DNA strands remained together in one of the daughter cells and the newly synthesised DNA strands went to the other daughter cell.

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The process of DNA replication in all organisms is amazing. The sum of all genes in a human cell 3 billion base pairs ,What's even more incredible is how few mistakes are made in this process despite the immense size of human DNA! An error occurs only about once in each 10100 billion bases. The complete process of DNA replication in human cells takes several hours. To replicate such huge molecules as human DNA at this speed requires not one, but many replication forks, forming replication bubbles and producing many segments of DNA strands that eventually meet up together and are joined to form the newly synthesized double helix.

Three fundamental processes take place in the transfer and use of genetic information : o Replication is the process by which a replica, or copy , of DNA is made .Replication occurs every time a cell divides so that information can be preserved and handed down to offspring. o Transcription : is the process by which the genetic messages contained in DNA are read or transcribed. The product of transcription , known as messenger RNA , leave the cell nucleus and carries the message to the site of protein synthesis. o Translation : is the process by which the genetic messages carried by mRNA are decoded and used to build proteins.

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Structure of RNA •

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There are three major types of RNA that participate in the process of protein synthesis: o ribosomal RNA , o transfer RNA and o messenger RNA. Like DNA , These three types of RNA are unbranched polymeric moleules composed of mononucleotides joined together by phosphatebonds. Most RNA exists as single strands .The three major types of RNA differ in size ,function and special structural modifications.

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Ribosomal RNA •

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Ribosomal RNA are found in association with several proteins as components of the ribosomes –the complex structures that serve as the site for protein synthesis. There are three distinct size species of r RNA ( 23S , 16S, and 5S ) in prokaryotic cells. In eukaryotic , there are four r RNA size species ( 28S , 18S , 5.8S , and 5S ) Note S is the Svedberg unit, which is related to the molecular weight and shape of the compound. R RNAs make up to 80% of the total RNA in the cell.

Transfer RNA •

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The smallest RNAs of the three major species of RNA molecules ( 4S) have between 74 and 95 nucleotides residues. There is at least one specific type of t RNA molecule for each of the twenty amino acids commonly found in proteins. tRNAs make up about 15 % of the total RNA in the cell. Each tRNA serves as an adaptor molecule that carries its specific amino acid attached to its 3” end to the site of protein synthesis. There it recognize the genetic code word on an mRNA , which specifies the addition of its amino acid to the growing peptide chain.

Messenger RNA • •

Messenger RNA comprises only about 5% of the RNA in the cell. The messenger RNA carries genetic information from the nuclear DNA to the cytosol , where it is used as the template for protein synthesis. Special structural of eukaryotic mRNA ( but not prokaryotic) include a long sequence of adenine nucleotides ( a poly-A tail ) on the 3” end of the RNA chain , plus a cap on the 5” end consisting of a moleule of 7-methylguanosine attached through a triphosphate linkage.

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DNA Repair • •

DNA is repaired if errors arise Some mechanisms involve removal of damaged area and synthesis of replacement

Types of Errors in DNA -

Deletion- nucleotide(s) removed Insertion- nucleotide(s) added Substitution- one nucleotide swapped for another o Transitions o Transversions o DNA modifications

Substitutions -

Bases can undergo tautomerization (movement of bonds) Uncommon but spontaneous Allows for mismatched nucleotide incorporation during synthesis

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DNA Modifications -

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Modified DNA is damaged DNA o Deamination of bases o Methylation of bases o Alkylation of bases o Inappropriate bonds created Different systems recognize and repair each

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Repair by Removal Nucleotide excision Distorted helix is seen Enzyme complex cuts bad strand on either side DNA polymerase I fills in gap DNA ligase seals patch in place

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Deamination of Cytosine: Repaired

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Direct Repair -

Damage is repaired in place o Ex: Thymine dimers and photolyase (E. coli) o UV damages DNA by causing crosslinking o Blocks replication o Repair involves breaking covalent bond

Why Care About Repair? -

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Xeroderma pigentosa o Damaged DNA repair system in skin o Death by multiple skin cancer by age 30 Hereditary nonpolyposis colon cancer o Defective mismatch repair

Repair of Damaged DNA -

Cancer, is the severe medically relevant consequence of the inability to repair damaged DNA.

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DNA damage can occur as the result of exposure to environmental stimuli such as alkylating chemicals or ultraviolet or radioactive irradiation and free radicals generated spontaneously in the oxidizing environment of the cell. These phenomena can lead to the introduction of mutations in the coding capacity of the DNA.

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Modification of the DNA bases by alkylation (predominately the incorporation of -CH3 groups) predominately occurs on purine residues. Methylation of G residues allows them to base pair with T instead of C. The protein itself becomes alkylated and is no longer active, thus, a single protein molecule can remove only one alkyl group.

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Mutations in DNA are of two types. Transition mutations result from the exchange of one purine, or pyrimidine, for another purine, or pyrimidine. Transversion mutations result from the exchange of a purine for a pyrimidine or visa versa.

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The prominent by-product from uvirradiation of DNA is the formation of thymine dimers. These form from two adjacent T residues in the DNA.

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Thymine dimers are removed by several mechanisms. Specific glycohydrolases recognize the dimer as abnormal and cleave the N-glycosidic bond of the bases in the dimer. This results in the base leaving and generates an apyrimidinic site in the DNA. This is repaired by DNA polymerase and ligase. Glycohydrolases are also responsible for the removal of other abnormal bases, not just thymine dimers.

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Humans defective in DNA repair, (in particular the repair of uv-induced thymine dimers), due to autosomal recessive genetic defects suffer from the disease Xeroderma pigmentosum (XP). There are at least nine distinct genetic defects associated with this disease. One of these is due to a defect in the gene coding for the glycohydrolase that cleaves the N-glycosidic bond of the thymine dimers. There are two major clinical forms of XP, one which leads to progressive degenerative changes in the eyes and skin and the other which also includes progressive neurological degeneration.

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Another inherited disorder affecting DNA repair in which patients suffer from sun sensitivity, and progressive neurological degeneration without an increased incidence of skin cancer is Cockayne Syndrome.

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(AT) is an autosomal recessive disorder resulting in neurological disability and suppressed immune function. Patients develop a disabling cerebellar ataxia early in life and have recurrent infections. Patients suffering from AT have an increased sensitivity to x-irradiation .

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Several diseases associated with defective repair of damaged DNA can be found in the Inborn Errors .

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Post-Replicative Modification of DNA, Methylation -

One of the major post-replicative reactions that modifies the DNA is methylation. The sites of natural methylation of eukaryotic DNA is always on cytosine residues that are present in CpG dinucleotides.

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Methylation of DNA in prokaryotic cells also occurs.

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The function of this methylation is to prevent degradation of host DNA in the presence of enzymatic activities synthesized by bacteria called restriction endonucleases. The role of this system in prokaryotic cells is to degrade invading viral DNAs. Since the viral DNAs are not modified by methylation they are degraded by the host restriction enzymes. The methylated host genome is resistant to the action of these enzymes.

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