Molecular Biology I BIO316 Lecture 3 PDF

Molecular Biology I BIO316 Lecture 3 Basic characteristics of DNA Replication Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 3 Basic characteristics of DNA Replication Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli DNA Replication Associate Professor of Biochemistry and Molecular Genetics  The accurate replication of DNA prior to cell division is a basic and crucial function.  This complex process requires the participation of multiple cellular proteins. Models of DNA replication  In replication, the sequence of parental strands must be duplicated in daughter strands.  One parental helix with two strands must yield two daughter helices with four strands.  Three models for DNA replication were evaluated in 1958 by Matthew Meselson and Franklin Stahl.  Dr. Rami Elshazli Models of DNA replication Associate Professor of Biochemistry and Molecular Genetics  Three models of DNA replication are possible:  Conservative model  Both strands of the parental duplex would remain intact (conserved).  The newly synthesized DNA strands consist of all-new molecules.  The conservative model produces one entirely new molecule and conserves the old.  Dr. Rami Elshazli Models of DNA replication Associate Professor of Biochemistry and Molecular Genetics  Three models of DNA replication are possible:  Semiconservative model  One strand of the parental duplex remains intact in daughter strands (semiconserved).  New complementary strand is built for each parental strand consisting of new molecules.  Daughter strands would consist of one parental strand and one newly synthesized strand.  Dr. Rami Elshazli Models of DNA replication Associate Professor of Biochemistry and Molecular Genetics  Three models of DNA replication are possible:  Dispersive model  The copies of DNA would consist of mixtures of parental and newly synthesized strands.  The dispersive model produces hybrid molecules with each strand a mixture of old and new.  Meselson and Stahl showed that the basic mechanism of replication is semiconservative.  Each new DNA helix is composed of one old strand and one new strand.  Requirements of DNA Replication  Replication requires three things:  Something to copy.  Something to do the copying.  The building blocks to make the copy.  The parental DNA molecules serve as a template.  Enzymes perform the actions of copying the template.  The building blocks are nucleoside triphosphates.  The replication process consists of three steps:  The beginning where the process starts.  The middle where the building blocks are added.  The end where the process is finished.  We could use three terms: initiation, elongation, and termination. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Action of DNA polymerase Associate Professor of Biochemistry and Molecular Genetics  DNA polymerase:  The enzyme that matches the existing DNA bases with complementary nucleotides and links the nucleotides together to make the new strand is called DNA polymerase.  DNA polymerases have several common features:  They add new bases to the 3′ end of existing strands.  They synthesize in a 5′-to-3′ direction by extending a strand base-paired to the template.  Each new base must be complementary to the base in the template strand.  All DNA polymerases also require a primer to begin synthesis.  RNA polymerases usually synthesize the primers.  The replication process requires a template to copy, nucleoside triphosphate building blocks, and the enzyme DNA polymerase.  DNA polymerases synthesize DNA in a 5′-to-3′ direction from a primer, usually RNA.  Dr. Rami Elshazli  Prokaryotic Replication Associate Professor of Biochemistry and Molecular Genetics  We would concentrate on prokaryotic replication using E. coli as a model.  Two separate replisomes are loaded onto the origin and  Replication in E. coli initiates at a specific site, the initiate synthesis in the opposite directions on the origin (called oriC), and ends at a specific site, the chromosome. terminus.  These two replisomes continue in opposite directions  After initiation, replication proceeds bidirectionally until they come to a unique termination site. from the unique origin to the unique terminus.  The DNA controlled by an origin is called a replicon.  Types of DNA polymerase  DNA polymerase refers to a class of enzymes that use a DNA template to assemble a new complementary strand.  DNA polymerase I (Pol I).  DNA polymerase II (Pol II).  DNA polymerase III (Pol III).  All three of these enzymes synthesize polynucleotide strands in the 5′-to-3′ direction and require a primer.  Function of DNA polymerases:  Nuclease enzymes:  DNA Polymerase I enzyme acts on the lagging strand to  Enzymes that act as nucleases are classified as: remove primers and replace them with DNA.  Endonucleases (cut DNA internally).  DNA Polymerase II enzyme does not appear to play a  Exonucleases (remove nucleotides from the end of DNA). role In replication but is involved in DNA repair  DNA polymerases have an exonuclease activity, which serves as a processes. proofreading function.  DNA Polymerase III is the main replication enzyme; it is  It allows the enzyme to remove a mispairing base. responsible for the bulk of DNA synthesis.  DNA Polymerase I has a 5′-to-3′ exonuclease activity, which used to Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics remove RNA primers.  Dr. Rami Elshazli Unwinding of DNA Associate Professor of Biochemistry and Molecular Genetics  Helicase enzyme:  Topoisomerases enzymes:  The enzyme with DNA unwinding activity is called a  Unwinding the helix produces torsional strain. helicase.  This can cause the double helix to further coil in space  This process requires energy in the form of ATP, and (supercoiling). can progressively unwind DNA, forming single  Topoisomerase enzymes act to relieve the strands. overwinding strain caused by unwinding and to  SSB Protein: prevent this supercoiling from happening.  These single strands are not stable because the  DNA gyrase is the topoisomerase involved in DNA hydrophobic bases are exposed to water. replication.  Cells solve this problem with another protein, single- strand-binding protein (SSB), that will coat exposed single strands.  Dr. Rami Elshazli Replication is semi-discontinuous Associate Professor of Biochemistry and Molecular Genetics  DNA has antiparallel nature, this meaning that one strand runs in the 3′-to-5′ direction, and its complementary strand runs in the 5′-to-3′ direction.  Because polymerases can synthesize DNA in only one direction, and the two DNA strands run in opposite directions.  Polymerases on the two strands must be synthesizing DNA in opposite directions.  The requirement of DNA polymerases for a primer means that on one strand primers need to be added as the helix is opened up.  The strand that is continuous is called the leading strand, and the strand that is discontinuous is the  The 5'-to-3’ synthesis of the polymerase and the lagging strand. antiparallel nature of DNA mean that only one strand,  DNA fragments synthesized on the lagging strand are the leading strand, can be synthesized continuously. named Okazaki fragments.  The other strand, the lagging strand, must be made in pieces, each with its own primer.  Dr. Rami Elshazli Replication is semi-discontinuous Associate Professor of Biochemistry and Molecular Genetics  Replication Fork Bacterial DNA Replication Proteins and Their Functions Protein Function  The unwinding process of a DNA helix to form two single Helicase Unwinds parental double helix at replication forks strands has a forked appearance and is thus called the Single-strand binding Binds to and stabilizes single-stranded replication fork. protein DNA until it is used as a template  This process requires the synthesis on leading strand Topoisomerase Relieves overwinding strain ahead of and lagging strand. (DNA gyrase) replication forks by breaking, swiveling, and rejoining DNA strands Primase Synthesizes an RNA primer at 5’ end of leading strand and at 5’ end of each Okazaki fragment of lagging strand DNA pol III - Using parental DNA as a template - Synthesizes new DNA strand by adding nucleotides to an RNA primer DNA pol I Removes RNA nucleotides of primer from 5’ end and replaces them with DNA nucleotides DNA ligase - Joins Okazaki fragments of lagging strand - on leading strand, joins 3’ end of DNA that replaces primer to rest of leading strand DNA Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  DNA primase Associate Professor of Biochemistry and Molecular Genetics  The primers required by DNA polymerases during replication are synthesized by the enzyme DNA primase.  This enzyme is an RNA polymerase that synthesizes short stretches of RNA (10 to 20) bp long that function as primers for DNA polymerase.  Leading strand  After synthesis of RNA primer, the leading strand can be elongated continuously by the action of DNA Pol III.  The Pol III enzyme is a large enzyme that has high processivity due to the action of one subunit of the enzyme, called the β subunit.  The β subunit is referred to as the “sliding clamp”.  For the clamp to function, it must be opened and then closed around the DNA.  A complex protein called the clamp loader accomplishes this task.  Dr. Rami Elshazli  Lagging strand Associate Professor of Biochemistry and Molecular Genetics  Because synthesis on the lagging strand is discontinuous, more steps are required to replicate this strand.  Primase is needed to synthesize RNA primers for each Okazaki fragment, and then all these RNA primers must be removed and replaced with DNA.  Finally, the fragments need to be stitched together.  The Okazaki fragments are synthesized by DNA Pol III.  DNA polymerase I:  The primers are removed and replaced with DNA by DNA Pol I.  With its 5′-to-3′ exonuclease activity, DNA Pol I can  The primers are removed by DNA polymerase I using its remove primers and then replace them. 5′-to-3′ exonuclease activity, then extending the  The DNA Pol I extends the Okazaki fragment “behind” previous Okazaki fragment to replace the RNA. it, while removing the RNA primer in “front” of it.  The nick between Okazaki fragments after primer removal is sealed by DNA ligase.   DNA replication by replisome Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The enzymes involved in DNA replication form a molecular assembly called the replisome.  This assembly forms the “replication organelle”.  The replisome has two main subcomponents:  The primosome.  Complex of two DNA Pol III enzymes, one for each strand.  The primosome consists of primase and helicase, along with various accessory proteins.  The two Pol III complexes include two synthetic core subunits, each with its own β sliding clamp subunit.  The entire replisome complex is held together by multiple proteins, including the clamp loader.  As DNA Pol III finishes an Okazaki fragment, the clamp loader loads a β subunit onto the next fragment and transfers the DNA Pol III to this new β subunit.  Dr. Rami Elshazli  DNA replication by replisome Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  DNA replication by replisome Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  DNA replication by replisome Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  DNA replication by replisome Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Replication is semi-discontinuous Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics