Replication Is Connected to the Cell Cycle Chapter 11 Replication Is Connected to the Cell Cycle Jocelyn E. Krebs

Figure 11. CO: A transmission electron micrograph of an E Figure 11.CO: A transmission electron micrograph of an E. coli bacterium in the early stages of binary fission, the process by which the bacterium divides. © CNRI/Photo Researchers, Inc.

11.1 Introduction Figure 11.01: A growing cell alternates between cell division of a mother cell into two daughter cells and growth back to the original size.

Figure 11.02: Replication initiates at the bacterial origin when a cell passes a critical threshold of size.

11.2 Bacterial Replication Is Connected to the Cell Cycle The doubling time of E. coli can vary over a 10x range, depending on growth conditions. It requires 40 minutes to replicate the bacterial chromosome (at normal temperature). Completion of a replication cycle triggers a bacterial division 20 minutes later.

Fast rates of growth therefore produce multiforked chromosomes. If the doubling time is ~60 minutes, a replication cycle is initiated before the division resulting from the previous replication cycle. Fast rates of growth therefore produce multiforked chromosomes. Figure 11.03: The fixed interval of 60 minutes between initiation of replication and cell division produces multiforked chromosomes in rapidly growing cells.

11.3 The Septum Divides a Bacterium into Progeny That Each Contain a Chromosome anucleate cell – Bacteria that lack a nucleoid but are of similar shape to wild-type bacteria. Bacterial chromosomes are specifically arranged and positioned inside cells. Septum formation is initiated mid-cell, 50% of the distance from the septum to each end of the bacterium.

Figure 11.04: Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.

The septum consists of the same peptidoglycans that comprise the bacterial envelope. The rod shape of E. coli is dependent on MreB, PBP2, and RodA. MreB: actin-like protein PBP2: transpeptidase RoA: SEDS, peptidoglycan formation FtsZ is necessary to recruit the enzymes needed to form the septum, peptidoglycan generation in septum. EnvA is required to split the covalent links between layers.

11.4 Mutations in Division or Segregation Affect Cell Shape fts mutants form long filaments because the septum that divides the daughter bacteria fails to form. Minicells form in mutants that produce too many septa. They are small and lack DNA. par mutation Anucleate cells of normal size are generated by partition mutants in which the duplicate chromosomes fail to separate.

fts mutants form long filaments Figure 11.05: Failure of cell division under nonpermissive temperatures generates multinucleated filaments. Photo courtesy of Sota Hiraga, Kyoto University.

11.5 FtsZ Is Necessary for Septum Formation The product of ftsZ is required for septum formation at pre-existing sites. Ox or knock out-septum formation defect FtsZ is tubulin-like protein and possess a GTPase activity that forms a ring (the Z-ring or septal ring) on the inside of the bacterial envelope. It is connected to other cytoskeletal components. Figure 11.07: Immunofluorescence with an antibody against FtsZ shows that it is localized at the mid-cell.

ZipA : integral membrane protein, located in the inner membrane ZipA and FtsA Both proteins are directly interaction with FtsZ , both proteins are required for septum formation. ZipA : integral membrane protein, located in the inner membrane FtsA; cytosolic protein FtsW: SEDS family (peptidoglycan formation) FtsI (PBP3) transpeptidase FtsW is responsible for FtsI localization at septum FtsZ is major cytoskeletal component of separatio in bacteria and chloroplast In Mito. dynamin Photo courtesy of William Margolin, University of Texas Medical School at Houston

11.6 min and noc/slm Genes Regulate the Location of the Septum The location of the septum is controlled by minC, -D, and –E and by noc/slm. The number and location of septa is determined by the ratio of MinE/MinC,D. Dynamic movement of the Min proteins in the cell sets up a pattern in which inhibition of Z-ring assembly is highest at the poles and lowest at midcell. Slm/Noc proteins prevent septation from occurring in the space occupied by the bacterial chromosome. SlmA is DNA binding protein, blocking the FtsZ

MinB locus deletion minicell Location of septum Min B lucus expresses Min C, D and E Min CD is FtsZ inhibitor (Min D activation requires Min C) Min D is ATPase, osilation FtsZ assembled at MinCD lower site (midcell) SlmA nucleoid occlusion; chromosome prevent FtsZ, Z-ring Figure 11.08: MinC/D is a division inhibitor whose action is confined to the poles by MinE.

11.7 Chromosomal Segregation May Require Site-Specific Recombination The Xer site-specific recombination system acts on a target sequence near the chromosome terminus. It recreates monomers if a generalized recombination event has converted the bacterial chromosome to a dimer. Figure 11.09: Intermolecular recombination merges monomers into dimers, and intramolecular recombination releases individual units from oligormers.

Figure 11.10: A circular chromosome replicates to produce two monomeric daughters that segregate to daughter cells.

Dif site: 28 bp target site of XerC and XerD; recombinase and located in termination region XerC binds to Dif and forms the holiday junction FtsK; septum located protein, large transmemb protein N-terminus; membrane association C-terminus; resolve Xer dimer to monomer and ATPase (translocation along DNA) First recombination site should be located within 30 Kb from Dif Figure 11.11: A recombination event creates two linked chromosomes. Xer creates a Holliday junction at the dif site, but can resolve it only in the presence of FtsK.

11.8 Partitioning Separates the Chromosomes Replicon origins are attached to the inner bacterial membrane. Chromosomes make abrupt movements from the midcenter to the one-quarter and three-quarter positions. Figure 11.12: The DNA of a single parental nucleoid becomes decondensed during replication. MukB is an essential component of the apparatus that recondenses the daughter nucleoids.

Chromosome separation is active process and requires protein synthesis Muk class mutation  anucleate phenotype mukA outer membrane protein (tolC), attached the chromosome at envelope mukB SMC; some phenotypes are suppressed by topA mutation (topoisomerase) mukE and F condensin

11.9 The Eukaryotic Growth Factor Signal Transduction Pathway signal transduction pathway - The process by which a stimulus or cellular state is sensed by and transmitted to pathways within the cell. oncogene - A gene that when mutated may cause cancer. The function of a growth factor is to cause dimerization of its receptor and subsequent phosphorylation of the cytoplasmic domain of the receptor.

The function of the growth factor receptor is to recruit the exchange factor SOS to the membrane to activate RAS. The function of activated RAS is to recruit RAF to the membrane to become activated. The function of RAF is to initiate a phosphorylation cascade leading to the phosphorylation of a set of transcription factors that can enter the nucleus and begin S phase.

Figure 11.13: Growth Factors and Growth Factor Receptors

Figure 11.14: CNK serves as a molecular scaffold platform for KSK-mediated RAF activation on the membrane after being recruited by active RAS-GTP. Adapted from Genes & Development 20, 807, 2006.

11.10 Checkpoint Control for Entry into S Phase: p53, A Guardian of the Checkpoint The tumor suppressor proteins p53 and Rb act as guardians of the cell. A set of serine/threonine protein kinases called cyclin-dependent kinases (CDKs) control cell cycle progression. Cyclin proteins are required to activate CDK proteins.

Figure 11. 15: Formation of an active CDK requires binding to a cyclin Figure 11.15: Formation of an active CDK requires binding to a cyclin. The process is regulated by positive and negative factors.

Figure 11.16: DNA damage pathway.

Figure 11.17: Growth factors are required to start the cell cycle and continue into S phase.

Figure 11.HP01: John Cairns and the Bacterial Chromosome