DNA REPLICATION

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DNA (Deoxyribonucleic acid) Site: Human DNA is present in the nucleus and mitochonria Function: carry genetic information. Structure: Human DNA consists of two strands of polydeoxyribonucleotides. The nucleotides are arranged in chains linked together by 3'→5′ phosphodiester bond between C3′ of deoxyribose of one nucleotide and OH of C5′ of the next one. phosphodiester bond means one phosphate is linked to 2 sugars. 1

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2 This is a tetranucleotide GCTA

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3 Each DNA strand has two ends; 5′ end ( the end with free phosphate group attached to C5′ of the first pentose) and 3′ end ( the end with free OH group on C3′ of the last pentose. The nucleotides in the polynucleotide chain is always read from 5′ → 3′ direction This part of polynucleotide is read ATC

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4 Base pairing: The two strands are linked together through hydrogen bonding formed between purine bases in one strand with pyrimidine bases in the other resulting in two types of base pairing: - Adenine is always paired with thtmine by 2 hydrogen bonds (A=T) - Guanine is always paired with cytosine by 3 H-bonds. _

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5 So in the double stranded DNA: 1- The content of adenine equals to that of thymine and the content of guanine is equal to that of cytosine 2- also, each strand must be complementary to the other i.e. each base of one strand is matched by a complementary hydrogen bonding base on the other strand.

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6 DNA double helix: The two strands of DNA wind arround each other forming double helix which stabilize DNA. Also abundance of hydrogen bonds between base help to stabilize DNA. Watson and Crick model

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8 Functions of DNA: 1- it is the storage site of genetic information. 2- DNA replication (reproduction): the stored information are transmitted from parent DNA to daughter DNA during cell division by a process called: replication. 3- transcription of mRNA for protein synthesis DNA replication ( DNA synthesis): DNAs have the ability to reproduce themselves by the process of replication, thus ensuring the transfer of genetic information from one generation to the next. DNA synthesizes replicate of itself by using its own structure as a template. Each strand of the double helix serves as a template for constructing a complementary daughter strand. The resulting double helix contains one parental and one daughter, and the mode of replication is thus called: Semiconservative

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9 Steps of DNA replication: occur in nucleus 1- Separation (unwinding) of double strands: Unpairing (separation, unwinding) of the two strands of the DNA double helix by: helicase enzyme with the formation of “replication fork ”. This separation is necessary because DNA polymerase III enzyme that responsible for replication use only one single stranded DNA as a template.

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10 This separation is maintained by a group of proteins includes: 1- Single stranded DNA-binding (SSB) proteins, also called: helix-destabilizing proteins: these bind to only single stranded DNA and keep two strands separated and prevent reformation of double helix. 2- DNA helicase: binds to single stranded DNA near the replication fork and then moves into the neighboring double-stranded region forcing the strands separation.

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11 2- Direction of replication: The enzyme responsible for replication is called: DNA polymerase III which forms a complementary copy from DNA template. DNA polymerase III read nucleotide sequence on DNA template from 3′ → 5′ direction so the synthesized new DNA strand will be antiparallel i.e in 5′ → 3′ direction. The 2 new strands grow in opposite direction, one in 5′ → 3′ direction toward replication and called “leading strand” which is synthesized continously. The other strand synthesized also in 5′ → 3′ direction but away from replication fork and called : “Lagging strand”.

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13 3-Starting synthesis of complementary strand by DNA polymerase III: This need RNA primer, because DNA polymerase III can’t join the first two nucleotides to start the new strand, instead it adds the nucleotides to the existing RNA primer. RNA primer is a short segment of RNA (8-10 nucleotides with free 3' OH end) consisting of RNA bases complementary to DNA sequence on the template. Primase: is the enzyme responsible for synthesis of RNA primer. Primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand without initial RNA primers. When DNA polymerase III recognizes RNA primer. It begins to synthesize new DNA strand using the 3' OH of RNA primer as the acceptor of the first deoxyribonucleotide.

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14 4- Removal of RNA primers by DNA polymerase I: When DNA polymerase III finishes synthesis of new DNA on lagging strand, RNA primers are removed by DNA polymerase I and the gap produced is filled by DNA synthesized by also DNA polymerase I. 5- DNA ligase: Connect (ligase) the newly synthesized DNA (Oazaki fragments) together

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17 Notes: 1- Leading strand is synthesized continuously so need only one RNA primer 2- Lagging strand is synthesized discontinuously so need many RNA primers. The newly DNA fragments formed are called Okazaki fragments 3- As a result of replication, 2 double stranded DNA is formed, each of which contain one old and one new strand, This is called: Semiconservative manner of replication.

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