Lec1 DNA Replication (1) PDF

College of Medicine Dr. Firas Al-Masoody First stage Biology Lecture 1 DNA Replication Learning objectives: 1. Describe the structure of DNA. 2. Explain the importance of DNA replication. 3. Explain t...

College of Medicine Dr. Firas Al-Masoody First stage Biology Lecture 1 DNA Replication Learning objectives: 1. Describe the structure of DNA. 2. Explain the importance of DNA replication. 3. Explain the process of DNA synthesis. 4. Identify the key enzymes involved in DNA replication and their functions. Introduction The structure of Deoxyribonucleic acid (DNA) was first described by James Watson and Francis Crick in 1953. DNA contains all the information necessary for the development and function of all organisms. DNA exists as a double helix, with about 10 nucleotide pairs per helical turn. Each of the two helical strands is composed of a sugar phosphate backbone with attached bases and is connected to a complementary strand by hydrogen bonding. The sugar in DNA is deoxyribose. The pairing of the nucleotide bases occurs such that adenine (A) binds with thymine (T) and guanine (G) with cytosine (C). Adenine forms two hydrogen bonds with thymine, while guanine and cytosine are connected by three hydrogen bonds. 1 Replication of DNA The replication or copying of cellular DNA is a necessary process to ensure that the instructions in DNA are faithfully passed on to the newly produced cells. The process of copying a DNA double helix is called DNA replication. When cells divide, each new cell gets an exact copy of DNA. During the S phase of interphase of cell cycle, when DNA is replicated, the double- stranded structure of DNA allows each original strand to serve as a template for the formation of a complementary new strand. In other words, if we designate the two DNA strands as S and S', strand S can serve as a template for making a new strand S’, while strand S' can serve as a template for making a new strand S. This results in the production of a new complementary strand that is identical to its former partner. The ability of each strand of a DNA molecule to act as a template for producing a complementary strand enables a cell to copy, or replicate, its genome before passing it on to its successors. Because replication can occur only on a single-stranded DNA template, the double-stranded DNA of the chromatin must first unwind. Once unwound, both strands of DNA are copied simultaneously. This process requires proteins to break open the double-stranded DNA, forming a replication fork. The main enzyme that catalyzes the formation of new DNA strands is DNA polymerase. 2  DNA replication is termed semiconservative, because each new double helix has one original strand and one new strand. In other words, one of the original strands is conserved, or present, in each new double helix. Each original strand has produced a new strand through complementary base pairing, so there are now two DNA helices identical to each other and to the original molecule. At the molecular level, several enzymes and proteins (known as replication machines) participate in the semiconservative replication of the new DNA strands. The process of DNA synthesis is started by initiator proteins that bind to specific DNA sequences called replication origin. As A-T base pair is held together by fewer hydrogen bonds than a G-C base pair, DNA rich in A-T base pairs is relatively easy to pull apart, and A-T- rich stretches of DNA are typically found at replication origins. The human genome has approximately 10,000 replication origins —an average of 220 origins per chromosome. At the replication origins, the initiator proteins force opens the two DNA strands apart, breaking the hydrogen bonds between the bases. Although the hydrogen bonds collectively make the DNA helix very stable, individually each hydrogen bond is weak. Once an initiator protein binds to DNA at a replication origin and locally opens up the double helix, it attracts the replication machine, in which each protein carries out a specific function in regard to DNA replication. Beginning DNA replication at many places at once greatly shortens the time a cell needs to copy its entire genome (8 hours in a rapidly dividing cell). Two replication forks (Y - shaped junctions) are formed at each replication origin. At each fork, a replication machine moves along the DNA, opening up the two strands of the double helix and using each strand as a template to make a new daughter strand. The two forks move away from the origin in opposite directions, unzipping the DNA double helix and replicating the DNA as they 3  go. DNA replication in eukaryotic chromosomes is therefore termed bidirectional. The major events in DNA replication include: 1. The enzyme DNA helicase unwinds double stranded DNA. 2. The initiator protein will join the DNA at replication origin and force open the two DNA strands apart by breaking the weak hydrogen bonds between the paired bases. 3. Short lengths of RNA act as primers for DNA synthesis: A specific enzyme called DNA primase, will join the exposed DNA strand to form short segment of RNA strand, known as RNA primer. RNA primer will form the starting point for the DNA polymerase enzyme. 4. New complementary DNA nucleotides, which are always present in the nucleus, are fit into place by the process of complementary base pairing. These are positioned and joined by the enzyme DNA polymerase. 5. Because the strands of DNA are oriented in an antiparallel configuration, and the DNA polymerase may add new nucleotides onto 3 end of the growing DNA strand (5 to 3* direction), DNA synthesis occurs in opposite directions. 6. To complete replication, the enzyme DNA ligase seals any breaks in the sugar- phosphate backbone. The DNA returns to its coiled structure. Characteristics of DNA synthesis A. Semiconservative with respect to parental strand When DNA is replicated during the process of cell division, one original strand of DNA is distributed to each daughter cell in combination with a newly synthesized strand. each of the two daughter strands has half new DNA and half old DNA; thus, the process is semiconservative. B. Bidirectional with multiple origins of replication Another characteristic of eukaryotic DNA replication is that it is bidirectional and starts in several different locations at once. DNA is copied at about 50 base pairs (bp} per second. 4  C. Primed by short stretches of RNA. A third characteristic of eukaryotic DNA replication is that it requires a short stretch of ribonucleic acid (RNA} for the initiation of the process. DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a single template. Instead, DNA primase, synthesizes short stretches of RNA that are complementary and antiparallel to the DNA template. The RNA primer is later removed. D. Semi-discontinuous with respect to the synthesis of new DNA A new strand of DNA is always synthesized in the 5' to 3' direction. Because the two strands of DNA are antiparallel, the strand being copied is read from the 3' end toward the 5' end. All DNA polymerases function in the same manner: They "read" a parental strand 3' to 5' and synthesize a complementary new strand 5' to 3'. Because parental DNA has two antiparallel strands, the DNA polymerase synthesizes one strand in the 5' to 3' continuously. This strand is called the leading strand. The continuous or leading strand is the one in which 5' to 3' synthesis proceeds in the same direction as replication fork movement. 5  The other new strand is synthesized 5' to 3', but discontinuously, creating fragments that ligate (Join) together later. This strand is called the discontinuous or lagging strand. The DNA synthesized on the lagging strand as short fragments (100 to 200 nucleotides) is called the Okazaki fragments. ENZYMES INVOLVED IN DNA SYNTHESIS Each step in the process of eukaryotic DNA replication requires the function of proteins (enzymes). Some of these proteins are described below. A. DNA polymerases: are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from one original DNA molecule. B. DNA helicases: Enzymes required to unwind short segments of the parental duplex DNA. C. DNA primases: DNA primases initiate the synthesis of an RNA molecule essential for priming DNA synthesis on both the leading and the lagging strands. D. Single-stranded DNA-binding proteins: prevent premature annealing of the single-stranded DNA to double-stranded DNA. They keep the strands protected until the complementary strands are produced. E. DNA ligase: DNA ligase is an enzyme that catalyzes the sealing of nicks (breaks) remaining in the DNA after DNA polymerase fills the gaps left by RNA primers. DNA ligase is required to create the final phosphodiester bond between the adjacent nucleotides on a strand of DNA. F. Telomerase: an enzyme that helps to maintain the telomere by adding TTAGGG repeats to the ends of the chromosomes. 6