DNA Synthesis

References:

Textbook, pages 224-252
Learning Objectives:
The chemical structure of DNA
The basic method of DNA replication in E. coli
including the rolling circle, theta and d-loop models
The DNA polymerases of eukaryotes that carry out DNA replication


As we all should know, the molecular basis of the traits described in classical genetics is DNA. DNA is copied (TRANSCRIBED) into RNA. RNA is a catalytically active molecule that participates in a number of reactions in the cell. As far as the traits analyzed in classical genetics is concerned, the RNA activity that is most important is protein synthesis, which is often called TRANSLATION because the sequence of nucleotides in an RNA is translated into a sequence of amino acids in a polypeptide. Polypeptides are processed into functional proteins in the cell. The activity of proteins in the cell, particularly the activity of protein enzymes, is responsible for the traits we observe in living organisms. The process of producing functional proteins from the information encoded in DNA is called GENE EXPRESSION. The branch of science that is focussed on the study of gene expression is MOLECULAR GENETICS. In our discussions of molecular genetics, we will focus on DNA synthesis, transcription and translation, with an emphasis on transcription, as the regulation of gene expression occurs mainly during transcription (the first step in gene expression).

DNA consists of 4 types of very similar organic molecules called deoxyNUCLEOTIDES. The precursors from which the chains of nucleotides (polynucleotides) are synthesized are deoxyNUCELOSIDE TRIPHOSPHATES (dNTP's).

dNTP's consist of a deoxyRIBOSE SUGAR, which has a base on its 1' carbon, and hydroxyl (-OH) on its 3' carbon and a triphosphate on its 5' carbon. The deoxyribose forms a ring resulting from the bonding of its 1' carbon to its 4' carbon (see figure 9.9 on page 227 of the textbook). All dNTPs are identical, except for the base. There are 4 bases found among dNTPs in cells:
 

ADENINE is found in deoxyadenosine triphosphate (dATP)
THYMINE is found in deoxythymidine triphosphate (dTTP)
CYTOSINE is found in deoxycytidine triphosphate (dCTP)
GUANINE is found in deoxyguanosine triphosphate (dGTP)
Adenine and guanine are PURINES, consisting of two rings, while  thymine and cytosine are PYRIMIDINES, consisting of a single ring. See figure 9.10 on page 227 of the textbook.

dNTPs are linked together by PHOSPHODIESTER BONDS which are formed by the reaction of a nucleoside triphosphate with the 3' carbon on the end of a nucleotide chain (see figure 9.11 on page 228 of the textbook). During the reaction, two of the phosphates are cleaved from the triphosphate of the dNTP, resulting in the incorporation of an deoxynucleoside triphosphate into the DNA chain ands the release of a pyrophosphate. This reaction is catalyzed by a class of protein enzymes called DNA POLYMERASES.

Since the nucleosides react at the 5' phosphate and the 3' hydroxyl of the deoxyribose sugar to form DNA strands, DNA molecules have a polarity with respect to DNA synthesis reactions. DNA molecules have a 5' END represented by the 5' phosphate group on the dNMP and a 3' END represented by the 3' hydroxyl group. DNA strands grow by the incorporation of a dNMP at the 3' end of the DNA strand. Thus DNA strands are said to grow in a 5' TO 3' direction.

The bases of the dNMPs (deoxynucleotide monophosphates) in DNA strands are paired via a COMPLIMENTARY interaction called HYDROGEN BONDING to form the DNA DOUBLE HELIX. Complimentarily is an important concept used to describe the interactions of biologically important molecules. Biological molecules interact because the fit together like a lock and key. The fit of nucleotides when the bases pair requires that the nucleotides be rotates 180o relative to each other (see figure 9.13 on page 230 of the textbook). The result of the geometry of base pairing is that the two DNA strands in a DNA double helix run in opposite directions.

Two bases hydrogen bonded to each other in a DNA double helix are called a BASE PAIR. Each base pair consists of one purine and one pyrimidine, causing the number of purines to equal the number of pyrimidines in a DNA double helix. Base pairs form between adenine and thymine (AT BASE PAIRS) and guanine and cytosine (GC BASE PAIRS). There are thus the same number of dAMPs and dTMPs in a DNA double helix and the same number of damps and damps in a DNA double helix. AT base pairs are held together by 2 hydrogen bonds and GC base pairs are held together by three hydrogen bonds, thus GC base pairs are held together more strongly than AT base pairs. See figure 9.14 on page 231 of the textbook.

The structure of the DNA double helix is due to the means by which DNA is replicated. During DNA synthesis there is localized separation of the double helix, with each strand serving as a TEMPLATE for the synthesis of a new complimentary strand. The result of DNA synthesis is the manufacture of two daughter DNA double helices from one parent double helix, with each daughter double helix consisting of one strand from the parent double helix (the template) and one newly synthesized strand. During DNA synthesis, the bases in the newly synthesized strands are determined by the matching of dNTPs to the dNMPs in the template strand using the base pairing rules described above (see figure 9.17 on page 232 of the textbook).

There are several different DNA polymerases in cells. The mechanism of DNA synthesis was first resolved for the prokaryote E. coli, where there are three different DNA polymerases, DNA POLYMERASE I, DNA POLYMERASE II, and DNA POLYMERASE III. In eukaryotic cells there are 5 different DNA polymerases, POLYMERASE a, POLYMERASE b, POLYMERASEg, POLYMERASE d, and POLYMERASE e. Polymerase g is located in mitochondria and is responsible for the replication of mitochondrial DNA. Note: the genes for all eukaryotic DNA polymerases are located on chromosomal DNA in the nucleus. Polymerase b is active in both dividing and non-dividing cells, and is thus thought to be responsible for DNA repair. The other three eukaryotic DNA polymerases, polymerase a, polymerase d, and polymerase e are active only in dividing cells, and thus are thought to be the DNA polymerases involved in replication.

We will focus specifically on the DNA replication system in E. coli, as it has been fully resolved. Eukaryotic DNA synthesis is highly similar.

When DNA is replicated, structures called REPLICATION FORKS appear in DNA molecules. At each replication fork, the parent DNA double helix is separated, with each strand then becoming a template for the synthesis of a new daughter strand (see figure 9.25 on page 239 of the textbook). Two strands are continuously synthesized at each replication fork. As the strands are synthesized, the replication fork moves down the DNA double helix.

The replication fork moves in one direction, synthesizing both strands. Given that the strands are ANTIPARALLEL (they run in opposite directions), the 5' to 3' direction of synthesis in one strand is the same direction that the replication fork moves and the 5' to 3' direction of synthesis of the other strand is opposite to the direction that the replication fork moves. As a consequence, the strand for with the synthesis is in the same direction as the movement of the replication fork, DNA synthesis is continuous, and this strand is called the LEADING STRAND. The strand for which synthesis is opposite the direction of synthesis, short fragments are made ( in the direction opposite to which the replication fork moves), and this strand is called the LAGGING STRAND. The short fragments found in the lagging strand are called OKAZAKI FRAGMENTS. See figure 9.27 on page 240 of the textbook.

DNA Replication

DNA polymerases add to the 3' end of existing polynucleotide strands. This poses a difficulty in DNA synthesis, as there is no 3' end following localized unwinding of the DNA double helix. To initiate DNA synthesis, a short strand of RNA (10 - 12 nucleotides long) called a PRIMER, which is made by an RNA polymerase called PRIMASE. Se figure 9.32 on page 246 of the textbook. DNA polymerase III then extends the RNA primer by adding dNTPs to the 3' end. Primers must be made repeatedly on the lagging strand. In all cases, the RNA primers must be removed. In E. coli, RNA primers are removed by a complex of RNASE H and DNA polymerase I. RNase H removes RNA from DNA/RNA hybrid molecules. Since it removes nucleotides from the end of a nucleic acid strand, DNA polymerase I is an EXONUCLEASE. Exonucleases can remove nucleotides from the 3' end (thus moving in a 3' to 5' direction, and called 3' to 5' exonucleases) or the 5' end (thus moving in a 5' to 3' direction, and called 5, to 3' exonucleases). RNA primers are extended in a 5' to 3' direction, and thus the primer begins at the 5' end of a fragment. The removal of the primer thus occurs in a 5' to 3' direction (see figure 5.6 on page 182 of Cooper). As the exonuclease activity of DNA polymerase I removes the primer, the DNA polymerase activity adds onto the 3' end of the neighboring fragment (see figure 9.29 on page 242 of the textbook). This activity of removing a NMP and replacing it with a dNMP causes the "gap" between fragments to move in a 5' to 3' direction. The "gap" is a missing phosphodiester bond between neighboring nucleotides which is often called a NICK. The movement of a nick as a consequence of the activity of DNA polymerase I in removing the RNA primer and replacing it with DNA is called NICK TRANSLATION. It is not clear what DNA polymerase II does in prokaryotes, as cells with the gene for DNA polymerase II deleted appear to function normally.

In eukaryotes, other exonucleases remove the RNA primers, and the gaps are filled by polymerase d. In all cases, nicks are sealed (the phosphodiester is made) by the enzyme DNA LIGASE, which requires the hydrolysis of ATP, as there are not dNTPs present at a nick. See figure 9.30 on page 243 of the textbook.
 

Nick Translation

A nick occurs when the phosphodiester bond between two nucleotides in one of the two strands of a DNA molecule is hydolyzed. At a nick, there will be a free phosphate at the 5' end of one nucleotide and a free -OH at the 3' end of another nucleotide. DNA polymerase I will add nucleotides to the 3' end and remove nucleotides from the 5' end. In doing so, DNA polymerase I will replace about 15 nucleotides, starting at the original nick and moving down in a 5' to 3' direction. The nick is also moved (translated) down about 15 nucleotides. DNA ligase then seals the nick. DNA ligase is an enzyne that will react with ATP at a nick to make a new phosphodiester bond. DNA polymerase cannot seal a nick as the nucleotide at the 5' end must have a triphosphate, but at a nick the 5' nucleotide has only a monophosphate.



 

The Okazaki fragments of the lagging strand and the leading strand are synthesized by DNA polymerase III in prokaryotes like E. coli. DNA polymerase I removes the RNA primers and replaces them with DNA. In eukaryotic cells, polymerase a is found in a complex with primase, and appears to be involved in the synthesis of short DNA/RNA fragments during lagging strand synthesis. Polymerase d then extends the short RNA/DNA fragments. Polymerase d also extends the leading strand. The role of ploymerase e is not clear.

Proteins other than DNA polymerases are involved in DNA replication. DNA polymerase III and polymerase d are associated with accessory proteins like the SLIDING CLAMP PROTEINS, that help the DNA polymerases to attach to the primers and maintain the stability of the DNA polymerase with the DNA. See figures 9.31 on page 245 of the textbook and 9.34 on page 247 of the textbook.

DNA replication requires the unwinding and separation of the DNA double helix, which is accomplished by enzymes called HELICASES. SINGLE STRANDED DNA BINDING PROTEINS then bind to the single standed DNA for stability and to prevent reassociation of the two DNA strands. See figure 9.32 on page 246 of the textbook. A complex of primase and helicase forms a macromolecular mchine called a PRIMISOME, which unwinds the DNA double helix and makes primers.

As the DNA is unwound, it creates a tension in the DNA in front of the replication fork, causing the DNA to rotate. To prevent  continuous rotation of the linear eukaryotic chromosomes and to prevent twisting of the circular prokaryotic chromosomes (see figure 9.38 on page 250 of the textbook), the tension is relieved by a class of enzymes called TOPOISOMERASES. Topoisomerase catalyze the breakage and rejoining of DNA strand. TYPE I topoisomerases introduce single nicks in one strand of the DNA double helix. TYPE II topoisomerases introduce nicks into both strands of the DNA double helix. Such breaks allow the DNA to rotate at the point of tension. Release of such tension prevents the tangling of linear eukaryotic chromosomes that would be caused by the constant rotation of the parental DNA double helix.

During DNA replication at the replication fork, the enzymes associated with the extension of the leading and lagging strand (DNA polymerase III in prokaryotes, polymerase d in eukaryotes, are in pairs at the replication fork (see figure 9.35 on page 248 of the textbook), with one of each pair directing replication of the leading strand and one of each pair directing replication of the lagging strand. The lagging strand is thought to form a loop such that each strand is synthesized in the direction of movement of the replication fork.

Note how long loops in the lagging strand are formed by the unwinding of the parent strand, and then copied by the REPLISOME COMPLEX. A replisome includes the primosome and DNA polymerase III complexes on both strands of DNA being copied. The copying of the lagging strand by DNA Pol III lags behind the unwinding of the lagging strand by helicase becaus the single stranded DNA has to wind around the replisome and orient itself in the proper (5' to 3') direction in the replisome. In the leading strand, the unwinding of the DNA helix immediatly preceeds the copying of the leadning strand by DNA Pol III,

The accuracy of DNA replication is not 100%. Mutations must occur. The rate of mutation, however, is critical to successful reproduction. If the rate of mutation is too great, and organism cannot successfully reproduce functioning copies of the genes needed for development. Rare tautomeric forms of nucleotides (nucleotides with alternate conformations) can be misincorporated, forming GT base pairs, for example at a rate of 1/10000 (one per per 104 bases). However, in the cell the rate is less than 1/1000000000 to 1/10000000000 (one in 109 to one in 1010 bases). DNA polymerases discriminate between correct and incorrect bases, apparently recognizing a correct base over an incorrect base, independent of the hydrogen bonding associated with base pairing. None-the-less, DNA polymerases will still incorporate an incorrect base about 1/1000000 (one in 106 bases).

DNA polymerases are also capable of PROOFREADING via 3' to 5' exonuclease activity (removal of nucleotides from the 3' end). Both prokaryotic DNA polymerase III and eukaryotic polymerase d have 3' to 5' exonuclease activity. Upon incorporation of a mismatched base (tautomers revert back to the more common conformation rapidly), the 3' to 5' exonuclease activity of DNA polymerase can remove them.

DNA replication begins at specific DNA sequences called ORIGINS OF REPLICATION, which are recognized by specific proteins that recruit the array of replication proteins. Plasmids, virus DNA and bacterial chromosomes have single origins of replication, the much larger eukaryotic genomes require multiple origins of replication in order to be replicated in a timely manner. Origins of replication, calledAUTONOMOUSLY REPLICATING SEQUENCES (ARS) have been isolated in yeast. ARSs consist of about 100 base pairs, containing a 11 bp sequence common to different kinds of ARSs. The proteins that associate with ARSs in yeasts are called the ORIGIN REPLICATION COMPLEX (ORC) and initiate DNA replication in yeasts. Yeast ORCs are very similar to those in other eukaryotes, including humans.

Telomeres

Because DNA is replicated only in the 5' to 3' direction, DNA polymerases cannot replicate the extreme 5' ends of linear chromosomes. Special mechanisms are thus required to replicate the ends of eukaryotic (linear) chromosomes. The sequences at the ends of eukaryotic chromosomes are called TELOMERES, and consist of tandem (side by side) repeats of a simple sequence of DNA. Telomeres are replicated by an enzyme complex called TELOMERASE, which catalyzes the synthesis of telomeres without a DNA template.

Telomerase is a type of REVERSE TRANSCRIPTASE, which synthesizes a DNA strand from an RNA template. Telomerases contain an RNA molecule that includes the template for the telomerase sequence. During eukaryotic DNA replication, telomerase binds to the ends of linear chromosomes by the complimentary base pairing of the template RNA in the telomerase to single stranded DNA at the end of the chromosome. The DNA strand at the end of the chromosome with the 3' end  (the leading strand) is then extended. The  DNA strand with the 5' end is then extended by conventional primase and DNA polymerase activity, and following the removal of the RNA primer, the telomere is extended. This process can be repeated many times. See figure 15.28 on page 421 and related text in the textbook.

Bacterial and viral DNAs are sometimes replicated using a method called the ROLLING CIRCLE MODEL of DNA replication. Usually, prokaryotic DNAs are replicated in both directions from an origin of replication, generally as shown in diagrams like figure 9.39 on page 251 of the textbook. However, some genomes, especially plasmids and viral genomes, are replicated by first making a nick in the DNA, with one replication fork moving around the circular genome as showm in figure 9.40 on page 252 of the textbook, producing a linear DNA that can be circularized by ligation of the ends, or it can remain linear. The rolling circle method allows for a single genome to be copied many times, producing a single very long linear DNA called a CONCATAMER in the case of viral DNA. The concatamer can then be cut into individual genomes and packaged.

Our textbook mentions D-loops and theta structures in it's descriptions of prokaryotic DNA replication. With D-loops, the replication of each of the strands begins at a different origin of replication. D-loop type replication occurs in chloroplasts and mitochondrial DNA. Theta structures result when the origin of replication for both strands of the circular DNA double helix begins at the same point. The result of D-loop replication is two separate circular DNA molecules following replication (see figure 9.41 on page 252 of the textbook). The result of theta structure type DNA replication is two strands looped together is something is not done to resolve the problem (this is the main reason for having topoisomerase type II).

Questions:

3, 6 (DNA is DS, RNS is SS), 7, 8, 14-17, on page 254-255 and question 15 on page 428 of the textbook.

References:

Textbook, pages 224-252 and 420-421.